1

Introduction

Sickle cell disease (SCD) is the most common hereditary hematological disorder throughout Africa, Middle East, and United States [ ]. SCD is characterized by chronic red blood cell hemolysis and vas occlusion associated with hypoxia [ ]. The pathophysiology of the disease is due to the polymerization of HbS in RBCs, leading to the occlusion of blood vessels [ ]. Children with SCD are at risk of severe morbidity that may impair their quality of life [ ]. Patients suffering from sickle cell anemia (SCA)had increased oxidative stress and peroxidation due to low antioxidants, leading to vaso-occlusive and painful crises [ ]. Impaired antioxidants status is due to reduced antioxidant defense mechanisms which indirectly depend on zinc micronutrient [ ]. Zinc is an essential trace element for all forms of life, also crucial for the division of cells and the synthesis of DNA and proteins [ ]. Zinc loss from biological membranes increases their susceptibility to oxidative damage and impairs their function [ ]. Significant effects of zinc deficiency include growth retardation, increased oxidative stress, and increased generation of inflammatory cytokines [ ]. Zinc has been considered an efficient antioxidant agent among humans [ , ]. Zinc inhibits lipid peroxidation activity in red blood cells and protects against oxidative stress, and subsequently drops of the incidence of vasocclusive and painful crisis [ ]. Researchers found that zinc deficiency is a common feature among SCA patients [ , ]. Zinc deficiency was connected with chronic hemolysis, increased demand and utilization, and secondary loss of zinc in urine [ , ]. Certain clinical features are shared between SCA patients and zinc-deficient patients, like delayed puberty, short stature, and low body weight [ ]. The amount of zinc in plasma, erythrocytes, and hair was lower, and the urinary excretion of zinc is higher in SCA patients than in healthy controls [ , , ]. Zn deficiency can be diagnosed by both clinical features and laboratory findings [ ].

Oral health is part of overall health and is an essential component of quality of life [ ]. Many systemic diseases affect oral and periodontal health [ ]. SCA is characterized by an impaired immune function that can affect oral health [ ]. Relation between salivary zinc and oral health revealed that zinc is essential for oral health maintenance [ ]. Earlier studies discovered that a low zinc diet profoundly influences oral health and dental caries formation [ ]. Several studies discussed the association between gingival and periodontal health and SCA status without conclusive results [ ]. There is no published articles that determined the level of zinc in the saliva of sickle patients. Advancements in treatment and medical care have allowed patients with SCA to have a better quality of life [ , ]. Therefore, it is essential to explore the impact of SCA on oral health and investigate possible underline mechanisms and pathogenesis.

Best to our knowledge, this is the first study investigated the salivary level of zinc and its relationship to the periodontal health of children and adolescents with SCA compared to healthy control groups.

2

Main Texts

A cross-sectional study was conducted at Khartoum state. The salivary samples were collected from thirty SCA cases regularly visiting the refer clinics of I The study was conduct in Khartoum state at different governmental hospital (IbrahiemMaliek teaching hospital and Jaferibnoof hospital) and thirty matched healthy subjects visiting Khartoum dental teaching hospital without any periodontal disease. The study was conducted for six months from January to June 2019. Participants included in the study were aged 5–18 years old in both cases and healthy control groups; known sickle cell anemic children regularly visit the refer clinic for follow-up. Patients with cardiovascular disease, epilepsy, and renal disorders and those with a history of antioxidant intake for the past three months were excluded. Clinical examination was evaluated with the inclusion criteria; they were informed about the study. After the parents agreed to participate, a questionnaire was obtained by the principal investigator asking about personal data and the history of the disease.

Stimulated whole saliva (2 ml) has been collected in the morning from participants between 9.00 and 11.00 a.m. The subjects have been asked to rinse the mouth with distilled water thoroughly to remove any food debris. The participants allowed the saliva to drool on the floor of the mouth until an adequate amount accumulated, then they were spitted off into a plain test tube. The procedure has been repeated until enough is collected.

3

Clinical oral examination and periodontal examination

The plaque index (PI; Silness and Loe) and gingival index(GI; Loe and Silness) were scored.The plaque index with PI = 0 means good OH, PI 1–2 fair OH, PI = 3 needy OH (23). The gingival index was scored; (GI = 0, no signs of gingival Inflammation, GI = 1 means sign of Inflammation without bleeding on probing, GI = 2, gingival Inflammation provoked bleeding) GI = 3 Spontaneous bleeding. Probing depth (PD) has been measured in millimeters with a Williams periodontal probe by the principal investigator using infection control equipment.

Score 0 = Normal gingiva; Score 1 = Gingival inflammation – a slight change in color, slight edema.

Oral hygiene was assessed utilizing Simplified Oral Hygiene Index based on Debris Index and Calculus Index.

4

Saliva sample preparation for zinc estimation

Frozen saliva samples have been allowed to dissolve at room temperature before their analysis and subjected to biochemical preparation and centrifugation for 15 min at 4000 rpm and diluted by 1% nitric acid 1:10(44). In the salivary sample solution, the element is subjected to high thermal energy to produce the excited state atom performed by the flame of Atomic Absorption Spectrophotometry.

Data were analyzed using SPSS version 23. Descriptive statistics (mean, standard deviation, minimum, and maximum) were used in the present study. Inferential statistics (Student t-test, p-value, and Spearman correlation) were also included. The significance level was accepted at P ≤ 0.05.

5

Results

The mean ages of subjects in the sickle cell anaemic group and control group were 10.37 ± 4.13years and 9.37 ± 3.6 years, respectively. Sickle cell anemic group composed of 14 (47%) Males and 16 (53%) Females and the control group composed of 16 (53%) Males, 14 (47%) Females ( Table 1 ) SCA patients had been diagnosed at an early age about 6 months. Regarding the past medical history, more than 80% of SCA patients developed early sign and symptoms of upper and lower limb swelling combined with severe pain. Concerning the management of the disease, all SCA patients were using folic acid while 19 and 5 took in addition, hydroxyurea and omega-three respectively. More than 80% of the patient have never visit dental clinic before; only 16% of them had history of extraction and restorative treatment, 86.7% had fair oral hygiene ( Table 2 ). There was a significantly lower level of salivary zinc among sickle cell anemic patients compared to healthy children with the salivary zinc level on an average of 0.5998 ± 0.116 mg/l in the control group and 0.18287 ± 0.103 mg/l in patients group (normal salivary level (0.5–1.2 mg/L) [ ]. Mean differences illustrated that the zinc level in the control group is on average 0.417 greater than the patient group (p < 0.0001) ( Fig. I ). Medication showed no effect on saliva zinc level ( Fig. II ). Zinc level in SCA patients with gingival inflammation were on average 0.08 less than SCA with normal periodontal health (P. Value: 0.025) ( Fig. III ).

Table 2

Dental history among sickle cell anemia group.

CHARACTERSTICS		Frequency	Percent %
had you visit a dental clinic	No	25	83.3
	Yes	5	16.7
	Total	30	100.0
Type of dentition	Primary	6	20.0
	Mixed	13	43.3
	Permanent	11	36.7
	Total	30	100.0
Periodontal health	Normal	14	46.7
	Gingival inflammation	16	53.3
	Total	30	100.0
Oral hygiene status	Poor	3	10.0
	Fair	26	86.7
	Total	29	96.7

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