Effects of a Honeybee Sting on the Serum Free Amino Acid Profile in Humans

<div><p>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.</p></div

Characteristics of the beekeepers involved in the study (n = 27).

<p>Characteristics of the beekeepers involved in the study (n = 27).</p

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

<p>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.</p

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).

<p>The explained variances are shown in brackets.</p

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

<p>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 < mean concentration in the serum of the beekeepers after at least six weeks since the last sting): Gln (d.value = –1.744), Met (d.value = –1.134).</p

Validation parameters of the LC-ESI-QqQ-MS/MS method used for determination of amino acids in serum of beekeepers.

<p>Validation parameters of the LC-ESI-QqQ-MS/MS method used for determination of amino acids in serum of beekeepers.</p

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

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

Session duration in NL and PL rats.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.</div

Percentage of rewards obtained (A-F), call rate (GH), and session duration (IJ) in rats grouped by performance in USV-training.

A. NL-0: Rats that did not obtain the maximum number of rewards in any training session (NL-0, non-learning “zero”, n = 34). B. NL-D1: Rats that obtained the maximum number of rewards on day 1 of training but not in the last 3 training sessions (NL-D1, non-learning “day one”, n = 17). C. NL-SGL: Rats that obtained the maximum number of rewards in only one training session, but not the first (NL-SGL, non-learning “single”, n = 5). D. NL-CEN: Rats that obtained the maximum number of rewards in a minimum of two consecutive training sessions, but not in the first two or last two (NL-CEN, non-learning” center”, n = 5). E. PL-PROG: Rats that obtained the maximum number of rewards during each of the last 3 training sessions, but not during other training sessions (PL-PROG, potentially learning “progress”, n = 10). F. PL-MAX: Rats that obtained the maximum number of rewards in all training sessions (PL-MAX, potentially learning “maximum”, n = 5). G. Call rate in rats from experiments with 7, 10, or 14 training sessions divided into six groups. H. Call rate in rats from experiments with 7, 10, or 14 training sessions divided into two groups: NL-SUM (all non-learning rats,) and PL-SUM (all potentially learning rats). Note that the call rate of PL-SUM rats is higher than that of NL-SUM rats in almost all training and test sessions. I. Session duration in all rats divided into six groups (as in G). J. Duration of a training session in PL-SUM and NL-SUM rats. Session time was shorter in PL-SUM rats than in NL-SUM rats from the second training session onward. The bars and line plots represent the mean ± SEM. The dots represent individual values for each rat. **p S3 and S4 Tables.</p

S2 Fig -

Changes in the percentage of rewards rats obtained and the duration of training sessions in operant-conditioning protocols 1–6 with vocalization emissions (A-I) or nosepokes (J-L) as rewarded responses. Rats could obtain a maximum of 10 or 30 rewards in one training session. A training session was terminated when a rat obtained the maximum number of rewards or the trial time exceeded 15 min. A. Protocol 1 (see also Fig 1 and Table 1 for protocols’ description): Food-restricted (95% initial body weight) rat was trained for 10 sessions, with a maximum of 10 rewards. B. Protocol 2: Conditions from Protocol 1 were modified by increasing food deprivation to 90% and extending training to 14 sessions. C. Protocol 3: Conditions from Protocol 2 were modified by adding 2 vocalization-eliciting stimuli: bedding from a cagemate and USV playback. D. Protocol 4: Conditions from Protocol 3 were modified by increasing the maximum number of rewards to 30. E. Protocol 5: Conditions from Protocol 4 were modified by performing training sessions in the dark phase instead of the light phase. F. Protocol 6: Conditions from Protocol 5 were modified by using habituation to the experimental cage (4 sessions before training) and reducing the training to 7 sessions. G. Protocol 6: Conditions were the same as in F, with habituation and training in a cage identical to the home cage. In all protocols, there was no overall increase in the number of rewards obtained (A, B, D, E, F), rather a decrease was observed (C: p H. Percent of rewards obtained during training from all protocols (1–6, A-G) pooled together; there was a decrease in the number of rewards (days 1–7: p S2b Table). I. Duration of training sessions from all protocols (1–6, A-G) showed no change in time (days 1–7: p = 0.7292, Friedman; p = 0.2324, Wilcoxon; days 1–10: p = 0.4746, Friedman; p = 0.7500, Wilcoxon; days 1–14: p = 0.9648, Friedman; p = 0.5412, Wilcoxon, S2b Table). J. Percent of rewards obtained by rats in nosepoke-conditioning. The majority of rats, initially trained in protocols 2 (n = 8), 5 (n = 8), and 6 (n = 20) obtained 100% of the rewards in the first nosepoke-training session (27/28 in the 30-reward protocol, 8/8 in the 10-reward protocol), which was maintained to the end of training. K. Duration of the nosepoke-training sessions decreased for both 10 (p S2c Table). L. Number of nosepokes during test sessions significantly decreased in subsequent test sessions for both maxima of 10 rewards (p = 0.0099, Friedman, p = 0.0391, Wilcoxon) and 30 rewards protocols (p S2d Table). The bars (A-H, J, L) and line plots (I, K) represent the mean ± SEM. The dots represent individual values for each rat. *p S2 Table. (TIF)</p