Circulating ghrelin rapidly responds to food intake, being decreased by carbohydrates and fat1 and increased by proteins1 or amino acids.2 Leptin also affects energy expenditure. While ghrelin stimulates food intake,3 leptin induces satiety.4 It has therefore been postulated as an antagonist of ghrelin.4
The salivary glands are additional sources of both peptides.5,6 Determination of salivary values has been suggested as a non‐invasive alternative to serum.7 We investigated the postprandial response of salivary ghrelin and leptin to determine whether this non‐invasive application is useful in clinical practice.
Serum and saliva were obtained from healthy males (n = 11; 18–40 years; body mass index (BMI) 19–27 kg/m2), before and two hours after intake of 1 g/kg glucose (fig 1). Patients (n = 18, 9.9 (4.4) years, BMI 13–26.5 kg/m2), underwent oral fructose (n = 9) or lactose tolerance tests (n = 9) because of suspected carbohydrate malabsorption. The sugar load was adjusted to 1 g/kg body mass. For ethical reasons only saliva was collected. Participants and parents gave informed consent.
Figure 1 Changes in serum and salivary ghrelin in healthy male adults (oral glucose tolerance test) (A) and in the saliva of children suspected of lactose/fructose malabsorption (B) before (0 min) and after (120 min) carbohydrate uptake (1 mg/kg body mass). The decline was present in all of the pre‐post samples in both environments (***p<0.001). No difference was found between either carbohydrate.
Ghrelin was measured by RIA (Mediagnost) with an LLOQ of 17 ng/l. Intra‐ and interassay coefficients of variation were 8.5% and 14%. Leptin was analysed by an inhouse RIA.8
Agreement between values obtained from matched serum and saliva was assessed by plotting the method differences against the method means.9 Differences between saliva and serum and between the two time points were calculated by the Wilcoxon signed rank test.
In the glucose group, a significant correlation between BMI and preprandial salivary and serum levels was found for ghrelin (r2 = 0.46; p<0.001) and leptin (r2 = 0.49; p<0.001). No comparable results were obtained for patients.
The mean absolute difference (%) between basal ghrelin in serum and saliva was −18.03 (30.51) (95% confidence interval −87.05 to 50.99). Values after glucose uptake yielded a mean absolute difference of −68.97 (15.48) (95% CI −104 to −33.95). Combination of pre‐and postprandial serum and saliva yielded absolute differences of −43.5 (78.9) (95% CI −80.43 to −6.56).
In the glucose group, initial ghrelin values ranged between 1.07 and 3.79 ng/ml (2.05 (1.03) ng/ml) in serum and 0.42 and 3.13 ng/ml (1.40 (0.90) ng/ml) in saliva. After glucose uptake these values decreased significantly (p<0.0001) in all participants by 25.9 (22.4)% in serum and 39.5 (28.1)% in saliva, yielding final concentrations of 0.62–3.13 ng/ml (1.47 (0.72) ng/ml) in serum and 0.33–2.11 ng/ml (0.76 (0.59) ng/ml) in saliva.
The same was found in patients after fructose or lactose uptake. Initial salivary ghrelin values ranged from 0.62 to 7.90 ng/ml (2.11 (1.85) ng/ml). After sugar intake, ghrelin decreased significantly (p<0.0001) by 45.2 (24.7)%, reaching final concentrations of 0.22–3.29 ng/ml (1.06 (0.86) ng/ml).
Several studies demonstrated a strong correlation between BMI and serum levels of both peptides, being positive for leptin and negative for ghrelin. In our study we found only a weak correlation between basal salivary values and BMI but this may have been due to the homogeneity of the participants' BMI values.
We found that leptin was not affected by carbohydrate uptake whereas ghrelin was decreased significantly in both environments two hours after sugar load. This was not surprising as leptin is a long term regulator of energy expenditure whereas ghrelin rapidly stimulates food intake in response to hunger.
In conclusion, we agree with Aydin and colleagues7 that salivary ghrelin may offer a non‐invasive alternatives to qualitative analysis, especially for dose‐response or kinetic studies. Yet there was no difference between healthy volunteers and patients with innocuous gastrointestinal disturbances. In ongoing studies we will investigate whether salivary ghrelin and leptin are potential diagnostic markers of gastrointestinal diseases.
Footnotes
Conflict of interest: None declared.
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