Effects of a honeybee sting on the serum free amino acid profile in humans

Abstract

The aim of this study was to assess the response to a honeybee venom by analyzing serum levels of 34 free amino acids. Another goal of this study was to apply complex analytic-bioinformatic-clinical strategy based on up-to-date achievements of mass spectrometry in metabolomic profiling. The amino acid profiles were determined using hybrid triple quadrupole/linear ion trap mass spectrometer coupled with a liquid chromatography instrument. Serum samples were collected from 27 beekeepers within 3 hours after they were stung and after a minimum of 6 weeks following the last sting. The differences in amino acid profiles were evaluated using MetaboAnalyst and ROCCET web portals. Chemometric tests showed statistically significant differences in the levels of L-glutamine (Gln), L-glutamic acid (Glu), L-methionine (Met) and 3-methyl-L-histidine (3MHis) between the two analyzed groups of serum samples. Gln and Glu appeared to be the most important metabolites for distinguishing the beekeepers tested shortly after a bee sting from those tested at least 6 weeks later. The role of some amino acids in the response of an organism to the honeybee sting was also discussed. This study indicated that proposed methodology may allow to identify the individuals just after the sting and those who were stung at least 6 weeks earlier. The results we obtained will contribute to better understanding of the human body response to the honeybee sting.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Extracted ion chromatograms of isobaric amino acids acquired during the analysis of one of the serum sample.

a: identified amino acids: 1-1MHis; 2-3MHis; b: identified amino acids: 1-Val; 2–Nval; c: identified amino acids: 1-Sar; 2-bAla; 3–Ala.

Figure 2. Three-dimensional (3D) partial least squares discriminant analysis separation using amino acids concentration-based metabolomics measurements in the serum of the beekeepers directly after a bee sting vs. the serum of the beekeepers after at least six weeks since the last sting (27 cases).

The explained variances are shown in brackets.

Figure 3. Variable importance in projection (VIP) plot: important features (analyzed serum free amino acids) identified by PLS-DA in a descending order of importance.

The graph represents relative contribution of amino acids to the variance between the stung and non-stung individuals. High value of VIP score indicates great contribution of the amino acids to the group separation. The black and white boxes on the right indicate whether the metabolite concentration is increased (black) or decreased (white) in the serum of the stung vs. non-stung beekeepers.

Figure 4. Significant features identified by SAM (Significance Analysis of Microarray (Delta = 0.3).

The more the variable deviates from the “observed-expected d” line, the more likely it is to be significant. The bold dots represent features that exceed the specified threshold (cutlow = –1.134, cutup = 1.155). Significant positive compounds (i.e. mean concentration in the serum of the beekeepers directly after a bee sting > mean concentration in the serum of the beekeepers after at least six weeks since the last sting): Glu (d.value = 2.717), 3MHis (d.value = 1.155). Significant negative compounds (i.e. mean concentration in the serum of the beekeepers directly after a bee sting

Figure 5

A: Receiver operating characteristic (ROC) curve for Gln. Cut-off value: 396.0 µM/l. Specificity: 90%, sensitivity: 90%. Area under the curve (AUC): 0.912, confidence interval: 95% (0.816–0.979); B: Concentration of Gln in two groups of beekeepers (directly after a sting and at least six weeks since the last sting). Dotted line indicates a cut-off value of 396.0 µM/l.