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\begin{document}

\title{Clinical significance of plasma PAF acetylhydrolase activity
measurements as a biomarker of anaphylaxis: cross-sectional study}



\author[1]{Krzysztof Piwowarek}%
\author[1]{Agnieszka Rzeszotarska}%
\author[1]{Jolanta Korsak}%
\author[1]{Aleksandra Juszkiewicz}%
\author[1]{Andrzej Chciałowski}%
\author[1]{Jerzy Kruszewski}%
\affil[1]{Military Institute of Medicine}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} Platelet-activating factor (PAF) has a direct role
as a mediator in the pathogenesis of various disorders with an
inflammatory component, including those with allergic aetiology. The
peripheral blood concentration of PAF is dynamically regulated by plasma
PAF acetylhydrolase (PAF-AH). Previous studies suggest that low activity
of plasma PAF-AH could be a predictive marker for increased severity of
some types of allergic hypersensitivity reactions -- especially
anaphylaxis. \textbf{Aim of the study:} The purpose of the study was to
evaluate the association between plasma PAF-AH activity and severity in
patients with anaphylactic reactions following a wasp or bee sting.
\textbf{Methods:} The study group of 89 patients was divided into two
subgroups depending on the increasing severity of the most severe
anaphylactic reaction in the past, which was assessed according to the
M\selectlanguage{ngerman}üller's scale. A control group of 20 people was established. Plasma
PAF-AH activity was measured using a colorimetric method.
\textbf{Results:} It has been observed that plasma activity of
platelet-activating factor acetylhydrolase was significantly lower in
patients with anaphylaxis history compared to the control group with
negative atopic history (on average 21.38 nmol/min/ml for the control
group, 9.47 nmol/min/ml for the first subgroup and 10.16 nmol/min/ml for
the second subgroup, in both cases p \textless{} 0.0001).
\textbf{Conclusion:} The plasma activity of PAF-AH is a promising
parameter that can help to distinguish a group of patients not
threatened with development of anaphylaxis and not requiring laborious
or expensive prophylactic procedures.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{1 \textbar{} INTRODUCTION}

Platelet-activating factor (PAF), which was discovered in 1972 {[}1{]},
is considered an important hypersensitivity mediator. Its influence
includes anaphylaxis and its most severe form -- anaphylactic shock. In
the murine BALB/c model it was found, that PAF together with histamine
may be responsible for the occurrence of anaphylaxis. The effect of PAF
was more related to the reduction of cardiac output than to generalized
vasodilation {[}2{]}. Moreover, some reports suggest that the importance
of PAF as a mediator of anaphylaxis in humans may be superior to
histamine and tryptase {[}3{]}.

The concentration of PAF in the peripheral blood is regulated by plasma
PAF acetylhydrolase (PAF-AH) {[}4, 5{]}. The physiological influence on
this enzyme seems to be multidirectional {[}6, 7{]}. Previous studies
indicate that the reduced activity of PAF-AH may be a potential risk
factor not only for the occurrence but also for the severe course of
food and \emph{Hymenoptera} venom allergies (HVA) {[}8-10{]}.

The aim of the study was to assess the relationship between the plasma
PAF-AH activity and the severity of anaphylactic reaction after honeybee
and wasp sting, towards a preliminary analysis of using this
relationship for diagnostic purposes.

\textbf{2 \textbar{} METHODS}

\textbf{2.1 \textbar{} Setting, study population, inclusion and
exclusion criteria}

The cross-sectional study was conducted in the Department of Infectious
Diseases and Allergology and in the outpatient allergology clinic of the
Military Institute of Medicine in Warsaw from December 2017 to April
2019.

All study participants had to be of legal age. The study group included
patients who registered in the clinic due to an anaphylactic episode of
varying severity after being stung by a honey bee or wasp. The episode
had occurred at least 21 days before enrolment in the study. The control
group consisted of healthy people with negative atopic history. The
exclusion criteria for both groups were identified as below:

1. The level of tryptase in the peripheral blood serum over 20 ng/ml.

2. History of congenital dyslipidemia.

3. History of mast cell dysfunction.

4. Acute disease.

\textbf{2.2.1 \textbar{} Patients and controls}

A group of 101 patients with confirmed IgE-dependent HVA were qualified
to participate in the study. Subsequently, 6 persons were disqualified
due to the presence of exclusion criteria.

The final study group of 45 women and 44 men, was then divided into two
subgroups: I-II, including participants with a history of
hypersensitivity reactions of maximum severity up to the second degree
according to the M\selectlanguage{ngerman}üller's scale {[}11{]} and III-IV, consisting of
patients who have experienced at least one grade III or IV reactions in
the past.

9 women and 11 men were qualified for the control group. They were
healthy and in a similar age range to the study group. They presented a
negative history of allergic and non-allergic hypersensitivity reactions
and did not meet the exclusion criteria.

\selectlanguage{english}\textbf{2.3 \textbar{} Ethical issues}

The study was performed according to the principles of the ICH-GCP
consolidated guidelines and the ethical principles regarding the current
revision of the Declaration of Helsinki together with Polish legal and
regulatory requirements {[}12{]}. Military Institute of Medicine
Bioethics Commission approval was obtained (number of decision:
58/WIM/2016).

\selectlanguage{english}\textbf{2.4 \textbar{} Data collection}

After obtaining informed consent, the researcher interviewed the
participants with a uniform questionnaire comprising of demographic and
other data necessary for proper group qualification. Subsequently, basic
anthropometric measurements were performed. Serum tryptase levels were
determined using the ImmunoCAP method.

Blood samples for the measurement of plasma PAF-AH activity were
collected on citrate and transported on ice to the laboratory.
Afterwards, the samples were centrifuged at 4 degC for 10 minutes at an
acceleration of 700 to 1000 g. The obtained plasma was stored frozen at
-80 degC. Finally, the authorized laboratory analyst defrosted samples and
loaded into the PAF Acetylhydrolase Assay Kit item No. 760901 produced
by Cayman Chemical Company (USA). The test, based on the colorimetric
method, was performed according to the manufacturer's instructions. We
used radiation with a wavelength of 412 nm. Measurements of the
absorbance were made six times in a row, at 1-minute intervals in order
to plot the appropriate linear function. The plasma PAF-AH activity was
calculated in units of nmol/min/ml using a properly modified formula
provided by the manufacturer of the kit.

\selectlanguage{english}\textbf{2.5 \textbar{} Statistical analysis}

All statistical tests were performed using the STATISTICA 13.0 software
provided by StatSoft Inc. (Poland). The Kolmogorov-Smirnow test was used
to determine the significance of differences in the distribution of the
studied variables in relation to the normal distribution. The t-test was
used for the comparison of means from the groups with a normal
distribution, while for others the Mann-Whitney U test was used. For
comparisons of multiple groups, the Bonferroni correction for the t-test
and Kruskal-Wallis ANOVA rank test were used, respectively.

Categorical variables were presented as n (\%) and compared with each
other using the \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} test. The control group and
research subgroups were paired to test the null hypothesis that plasma
PAF-AH activity is not related to the severity of the post-stinging
reaction. The established level of statistical significance was \selectlanguage{greek}α \selectlanguage{english}=
0.05.

The correlations between the variables were investigated by calculating
the Spearman's rank coefficient. It was considered significant when: r
\textgreater{} 0.3 or r \textless{} -0.3.

A regression analysis was performed and the receiver operating
characteristic (ROC) curve was laid out. Typical test parameters were
calculated according to well-known definitions, such as sensitivity,
specificity, accuracy, positive predictive value and negative predictive
value {[}13{]}.

\textbf{3 \textbar{} RESULTS}

\textbf{3.1 \textbar{} Population characteristics}

As shown in table 1 and 2, subgroups I-II included 8 patients with a
history of the first-degree reaction and 31 patients after second-degree
reactions. Subgroups III-IV included 33 patients after third-degree
reactions and 17 patients after fourth-degree reactions, respectively.

No statistically significant differences were found between subgroups
I-II, III-IV and the control group in terms of basic demographic,
anthropometric and biochemical data. In the studied subgroups,
IgE-dependent reaction to wasp venom was a more frequent cause of
post-stinging reactions (84.62\% in subgroups I-II and 80 \% in
subgroups III-IV, p = 0.98) than allergy to honey bee venom (15.38\% in
groups I-II and 20 \% in groups III-IV, p = 0.49); however, these
differences were statistically insignificant.

\textbf{3.2 \textbar{} Plasma PAF-AH activity}

The distributions of PAF-AH activity in the study group and the control
group were presented in figure 1. They differed substantially, which was
reflected in the highly statistically significant difference in the
arithmetic means in both groups.

As shown in figure 2, no significant differences were found in the
arithmetic mean values of plasma PAF-AH activity in the individual
degrees of severity of anaphylaxis (p \textgreater{} 0.05). Although,
these values were considerably lower than the value in the control
group, which was 21.38 \selectlanguage{ngerman}± 9.50 nmol/min/ml (p \textless{} 0.0001).

According to table 2, the arithmetic mean values \selectlanguage{english}\selectlanguage{english}of plasma PAF-AH
activity in the subgroup I-II (9.47 +- 1.87 nmol / min / ml) and in the
subgroup III-IV (10.16 +- 2.50 nmol/min/ml) also did not differ
significantly (p \textgreater{} 0.05), but were substantially lower as
compared to the value in the control group (p \textless{} 0.0001).

The arithmetic mean values \selectlanguage{english}\selectlanguage{english}of plasma PAF-AH activity in the smokers
and non-smokers did not differ significantly (9.46 +- 1.95 nmol/min/ml vs
10.13 +- 1.61 nmol/min/ml, p = 0.201).

There was no statistically significant correlation between the plasma
PAF-AH activity and most of the parameters, including the age of the
patients (r = -0.035). There was only a statistically significant, but
clinically irrelevant, correlation with tryptase concentration (r =
0.21).

\selectlanguage{english}\textbf{3.3 \textbar{} The performance of PAF-AH activity measurements
in recognizing anaphylaxis}

The result of the regression analysis is shown in figure 3. The optimal
cut-off point for PAF-AH activity to discriminate anaphylactic and
non-anaphylactic patients was 17.24 nmol/min/ml at the following
parameters:

\begin{itemize}
\tightlist
\item
  Sensitivity: 100\%
\item
  Specificity: 60\%
\item
  Accuracy: 93\%
\item
  Positive predictive value (PPV): 92.2\%
\item
  Negative predictive value (NPV): 100\%
\item
  Misclassification rate: 7\%
\item
  Area under the curve (AUC): 0.854
\item
  Slope factor: 0.21
\end{itemize}

\textbf{4 \textbar{} DISCUSSION}

In the presented study it was confirmed that the mean activity of plasma
PAF-AH activity is significantly lower in patients with a history of
anaphylaxis in the course of HVA as compared to healthy people. It
confirms the vital role of PAF in the pathogenesis of anaphylactic
reactions. Assuming that the plasma PAF-AH activity illustrates the
systemic activity of this enzyme, this fact indicates that apart from
external factors, the individual's specific predisposition resulting
from the enzyme activity may also play an important role.

Contrary to other authors, our research did not show an obvious
correlation between PAF-AH activity and the severity of the
hypersensitivity reaction to the venom {[}10{]}. This problem has been
studied and discussed since the early 2000s. Vadas et al. analyzed the
relationship between plasma PAF-AH activity and the severity of
anaphylactic reaction after peanut consumption in children {[}8-9,
14{]}. In the prospective part of the mentioned authors study, three
groups with increasing severity of anaphylaxis were recruited. The
control was a group of non-allergic children. In contrast to our study,
it showed a trend suggesting that decreased PAF-AH activity is
associated with a more severe course of an anaphylactic reaction.
However, statistical significance was not obtained.

A statistically significant association was found in the retrospective
section of the study. It compared 9 cases of fatal peanut anaphylactic
reactions to several other groups, including children who died from
non-anaphylactic causes, non-allergic children, and children with
urticaria or peanut-induced angioedema. In all these comparisons, the
plasma PAF-AH activity was significantly lower in children who died from
anaphylaxis. The study did not show any differences in PAF-AH activity
in groups of children with bronchial asthma of varying severity {[}8,
9{]}.

In subsequent years, the same researchers found that patients with the
lowest levels of plasma PAF-AH activity had a 27-fold higher risk of
severe or fatal anaphylaxis. Moreover, the plasma activity of the enzyme
was strongly correlated with the concentration of apolipoprotein B. It
is noteworthy that the activity of PAF-AH is also correlated with the
concentration of LDL and decreases during treatment with lovastatin or
fenofibrate. Therefore, it cannot be ruled out that lipid-lowering
therapy and lowering PAF-AH activity may lead to the increased risk of a
severe anaphylactic reaction in a susceptible population {[}14{]}.
Finally, Vadas et al. found a correlation between PAF-AH activity and
the age of the participants, but in our study, it was lacking.

Research on the plasma PAF-AH and food anaphylaxis was also conducted in
the Moscow study groups of children with a history of food anaphylaxis,
atopic dermatitis and a negative history of allergic diseases. It was
found that children with a history of food anaphylaxis had statistically
lowered level of PAF-AH mRNA in peripheral blood compared to both other
groups of children. In the group with atopic dermatitis, the difference
was more than three times. In the group of children with a history of
food anaphylaxis, lower expression of the PAF-AH gene was associated
with a more severe course of anaphylaxis and cardiovascular symptoms
{[}15{]}.

A large study on the relationship between plasma PAF-AH activity and the
severity of anaphylactic reactions to \emph{Hymenoptera} venom was
carried out in a Milan (Italy). Its structure was relatively similar to
our trial. There was no statistically significant difference in PAF-AH
activity between healthy controls and patients after the mild
anaphylactic reactions of the 1\textsuperscript{st} degree of severity.
The subgroups with higher severity grades had significantly and stepwise
lower mean plasma PAF-AH activity compared to the control group and to
each other. The exception has occurred for the 3rd and 4th severity
subgroups, between which the difference was statistically irrelevant.
Logistic regression showed that only PAF-AH activity was significantly
associated with the severity of anaphylaxis. The best cut-off point for
grades II-IV was 25 nmol/ml/min {[}10{]}.

The outcomes of our trial differ from the results of the presented
studies. The difference in the mean activity of the enzyme between the
control group and the study subgroups was more than twofold. The highest
PAF-AH activity in a patient with a positive history of anaphylaxis was
only 17.24 nmol/min/ml. The cut-off point of 20 nmol/min/mL proposed by
Pravettoni et al. discriminates patients who are not at risk of
developing severe anaphylaxis very well, but our study suggests the
possibility of further lowering this threshold. Notwithstanding, the
most intriguing is the combination of two study subgroups - grade I-II
and III-IV. They did not differ in terms of enzyme activity (9.47 vs
10.16 nmol/min/ml, p = 0.0609 in Student's t-test). Therefore, we did
not find a relationship between the severity of anaphylactic reaction in
the past and the plasma PAF-AH activity. This is interesting in the
context of the studies by Pravettoni et al., which show this kind of
association.

There are several potential reasons for the above-mentioned divergence.
Apart from the various size of the samples, age of the participants and
distinct local practice in qualifying patients for the different
severity grades of anaphylaxis, the discussed studies differed in
design. Unlike the first Vadas studies, it seems that our study group
comprised patients with less severe reactions, e.g. we did not
investigate cases of fatal anaphylaxis. The study performed by
Pravettoni et al. concerned only newly diagnosed patients, while our
study did not exclude patients undergoing immunotherapy. Technical
aspects affecting the results may also be important. In the Italian
study, 405 nm light was used to measure absorbance, while in our trial
wavelength was 412 nm. The dilution of samples could also be different;
however, to the extent permitted by the manufacturer of the assay kit.

Irrespective of the presented explanations, the results of our study
confirming the participation of PAF-AH in the pathogenesis of
anaphylaxis lead to the consideration of the practical application of
the measurements of plasma PAF-AH. In our population, all patients with
a history of anaphylaxis had decreased PAF-AH activity. This confirms
that the determination of PAF-AH activity can be a particularly useful
tool in the stratification of the individual risk of anaphylaxis
to\emph{Hymenoptera} venoms, especially in beekeepers. Assuming that the
clinical effect of stinging, on the one hand, depends on the strength of
the anaphylactic effect of the venom, and on the other hand on the
individual properties of the sensitized person, the measurement of
PAF-AH plasma activity allows us to assess a significant internal factor
influencing the occurrence of symptoms. Perhaps the test could also help
in the diagnosis of non-allergic and idiopathic anaphylaxis ---
distinguishing the true anaphylaxis from numerous conditions with
similar symptoms, such as somatoform disorders. Presently, the only
laboratory test widely used in such cases is the measurement of tryptase
concentration during an acute episode, which is often impossible to
perform for obvious reasons. The measurement of plasma PAF-AH activity
could provide a lot of diagnostic information, even months or years
after an anaphylaxis episode.

Unfortunately, our study plan did not take into account the group of
patients with other severe forms of hypersensitivity without a history
of an anaphylactic reaction. This would allow us to answer whether the
decreased PAF-AH activity occurs only in patients with anaphylaxis, or
in general in patients with severe hypersensitivity to one or more
factors.

A relatively wide dispersion of the results of plasma PAF-AH activity
was found in the control group. Noteworthy, this group was selected
primarily on the basis of negative atopic history. It is impossible to
predict a priori whether some of these people will be at risk of
developing anaphylaxis in the future. Perhaps currently healthy
individuals with low PAF-AH activity could be considered capable of
developing an anaphylactic reaction in the future. The distribution of
results in the control group may also suggest the presence of PAF-AH
gene polymorphisms in the Polish population, which strongly affect the
enzyme activity. These problems will be the subject of further research.

\textbf{5 \textbar{} CONCLUSIONS}

\begin{itemize}
\tightlist
\item
  The plasma activity of PAF-AH is a promising parameter that can help
  to distinguish a group of patients not threatened with development of
  anaphylaxis and not requiring laborious or expensive prophylactic
  procedures.
\item
  Reduced plasma PAF-AH activity below 17 nmol/min/ml is characteristic
  of patients who develop anaphylactic reactions after an insect sting.
\item
  Contrary to expectations, no significant correlation was found between
  the degree of impairment of plasma PAF-AH activity and the degree of
  severity of anaphylaxis following an insect sting.
\end{itemize}

\textbf{6 \textbar{} REFERENCES}

\begin{enumerate}
\tightlist
\item
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\item
  Wang M, Shibamoto T, Tanida M, et al. Mouse anaphylactic shock is
  caused by reduced cardiac output, but not by systemic vasodilatation
  or pulmonary vasoconstriction, via PAF and histamine\emph{. Life Sci}
  . 2014;116(2):98-105.
\item
  Vadas P, Perelman B, Liss G. Platelet-activating factor, histamine,
  and tryptase levels in human anaphylaxis. \emph{J Allergy Clin
  Immunol} . 2013;131(1):144-9.
\item
  Farr RS, Cox CP, Wardlow ML, et al. Preliminary studies of an
  acid-labile factor (ALF) in human sera that inactivates
  platelet-activating factor (PAF). \emph{Clin Immunol Immunopathol} .
  1980;15(3):318-30.
\item
  Stafforini DM, McIntyre TM, Carter ME et al. Human plasma
  platelet-activating factor acetylhydrolase. Association with
  lipoprotein particles and role in the degradation of
  platelet-activating factor. \emph{J Biol Chem} . 1987;262(9):4215-22.
\item
  McIntyre TM, Prescott SM, Stafforini DM. The emerging roles of PAF
  acetylhydrolase\emph{. J Lipid Res} . 2009;50 Suppl: S255-9.
\item
  Thompson A, Gao P, Orfei L, et al. Lipoprotein-associated
  phospholipase A\textsubscript{(2)} and risk of coronary disease,
  stroke, and mortality: collaborative analysis of 32 prospective
  studies. \emph{Lancet} . 2010;375(9725):1536-44.
\item
  Vadas P, Gold M, Liss G et al. PAF acetylhydrolase predisposes to
  fatal anaphylaxis. \emph{J Allergy Clin Immunol} . 2003;111(S206).
\item
  Vadas P, Gold M, Perelman B et al. Platelet-activating factor, PAF
  acetylhydrolase, and severe anaphylaxis. \emph{N Engl J Med} .
  2008;358(1):28-35.
\item
  Pravettoni V, Piantanida M, Primavesi L, et al. Basal
  platelet-activating factor acetylhydrolase: prognostic marker of
  severe Hymenoptera venom anaphylaxis. \emph{J Allergy Clin Immunol} .
  2014;133(4):1218-20.
\item
  Mueller HL. Diagnosis and treatment of insect sensitivity. \emph{J
  Asthma Res} . 1966;3(4):331-3.
\item
  World Medical Association. World Medical Association Declaration of
  Helsinki Ethical Principles for Medical Research Involving Human
  Subjects. \emph{JAMA} . 2013;310 (20), 2191-2194.
\item
  Florkowski CM. Sensitivity, Specificity, Receiver-Operating
  Characteristic (ROC) Curves and Likelihood Ratios: Communicating the
  Performance of Diagnostic Tests. \emph{Clin Biochem Rev} . 2008 Aug;
  29(Suppl 1): S83--S87.
\item
  Perelman B, Adil A, Vadas P. Relationship between platelet activating
  factor acetylhydrolase activity and apolipoprotein B levels in
  patients with peanut allergy. \emph{Allergy Asthma Clin
  Immunol.}2014;10(1):20.
\item
  Esakova N, Saakyan E, Kondratieva N i wsp.. Platelet-Activating
  Factor-Acetylhydrolase Gene (PLA2G7) Expression in Children with a
  History of Food Anaphylaxis. \emph{J Allergy Ther} . 2015;6(211).
\end{enumerate}

\textbf{Table 1.} Characteristics of the study subgroups - continuous
variables.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Variable}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup I-II} \textbf{n = 39}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup I-II} \textbf{n = 39}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup I-II} \textbf{n = 39}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup III-IV} \textbf{n = 50}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup III-IV} \textbf{n = 50}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Subgroup III-IV} \textbf{n = 50}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Control group} \textbf{n = 20}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Control group} \textbf{n = 20}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Control group} \textbf{n = 20}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Median}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Median}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Median}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{vs 1}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{vs 2}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{Age} \textbf{{[}years{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{48,26}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12,20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
46,00\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{45,36}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
13,88\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
43,50\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0,2301\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textbf{44,15}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
13,33\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
45,00\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0,2810\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0,9950\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Body mass {[}kg{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{81,95}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
17,99\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
85,00\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{76,56}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
15,16\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
75,00\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,1160\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{79,50}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
12,74\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
80,00\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,6637\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,4181\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Height {[}cm{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{171,86}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
9,86\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
171,00\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{171,02}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
9,09\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
170,00\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,5700\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{175,44}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
11,49\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
174,50\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,3528\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,1907\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{BMI {[}kg/m\textsuperscript{2}{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{27,59}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
5,03\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
27,14\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{26,05}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
4,01\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
25,46\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,1002\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{25,84}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
3,29\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
26,44\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,3023\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,7725\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Tryptase {[}ng/ml{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{5,59}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
4,13\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
3,94\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{4,77}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2,69\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
4,04\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,8167\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{5,46}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2,53\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
4,89\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,2878\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0,1465\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Tab. 2\emph{.}} Comparison of subgroups within the study
population regarding plasma PAF acetylhydrolase activity.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Comparised series}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Mean} \textbf{{[}nmol/min/ml{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Median}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Test}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Control group}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
21,38\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25,59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
U Manna-Whitney\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0,000001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Subgroup I-II}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,47\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1,87\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Control group}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
21,38\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25,59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
U Manna-Whitney\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0,000014\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Subgroup III-IV}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10,16\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2,50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,85\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Subgroup I-II}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,47\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1,87\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
t Student\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0,060902\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Subgroup III-IV}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10,16\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2,50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,85\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Control group}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
21,38\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25,59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Kruskal-Wallis test\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0,0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{1st degree of severity}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8,93\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2,13\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8,97\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{2nd degree of severity}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,61\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1,80\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,23\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{3rd degree of severity}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10,24\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2,64\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,67\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{4th degree of severity}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10,01\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2,26\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9,94\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\par\null

\textbf{Figure 1.} Scatter plot of PAF-AH plasma activity by severity of
anaphylaxis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2.} Distribution of PAF-AH plasma activity by severity of
anaphylaxis -- box plot. 0 -- control group, 1, 2, 3, 4 -- grade of
severity according to M\selectlanguage{ngerman}üller's classification.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 3.} ROC for PAF-AH plasma activity test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

