This appendix has been provided by the authors to give readers additional information about their work.
Supplement to: Vadas P, Gold M, Perelman B, et al. Platelet-activating factor, PAF acetylhydrolase, and severe
anaphylaxis. N Engl J Med 2008;358:28-35.
Radiolabeled PAF (1-O-hexadecyl-[2acetyl- 3H]-sn-glycero-3-phosphoholine,
499.5 Gbq/mmol) was purchased from NEN Life Science Products (Boston, MA).
Unlabeled PAF and lyso-PAF, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-
phosphocholine, egg yolk phosphatidylcholine, pefabloc (4-[2-
aminoethyl]benzenesulfonyl fluoride) were obtained from Sigma (Sigma-Aldrich
Canada, Oakville, Canada). Bond-Elute C18 columns were from Varian Inc.
Platelet activating factor 3H scintillation proximity assay (SPA) system and liquid
scintillation fluid (ACS) were supplied by Amersham Biosciences (Oakville,
Canada). Pre-Coated TLC Plates SILICA GEL 60 (layer thickness 0.25 mm, 20 x
20 cm) were purchased from Merck (Damstadt, Germany). All other chemicals
were from Sigma.
Measurement of Platelet Activating Factor in Human Blood
Blood samples to be assayed for PAF were treated as recommended by NEN
Life Sciences (Boston, MA). One volume of blood was vigorously shaken with an
equal volume of 20% acetic acid to inactivate PAF acetylhydrolase. Two
volumes of 10% acetic acid were added and the suspension was shaken again.
After centrifugation (12 000g, 60 min) the supernatant was recovered and the
pellet was subjected to a second extraction with 10% acetic acid in a similar
manner. These extracts were combined.
PAF was initially purified by chromatography on C-18 Octadecyl mini-columns
(200 mg of sorbent), according to the manufacturer’s instructions. Further
purification of the extracted PAF was performed by the method of Bligh and Dyer
with a mixture of chloroform-methanol-water (1:1:0.9) (1). The lower chloroform
phase, containing PAF, was removed and evaporated to dryness under a gentle
stream of nitrogen. Before assay, PAF was reconstituted with assay buffer by
vortexing for 15 min.
The concentration of PAF in samples was measured using the Platelet Activating
Factor 3H -Scintillation Proximity Assay (SPA) system (Amersham Biosciences,
Oakville, Canada). PAF was measured in the range of 0.2 - 12.8 ng/ml. Assay
specificity and sensitivity were determined by addition of known amounts of PAF
and subjecting each concentration to the extraction and purification procedures,
followed by SPA. The assay was sensitive to 20-25 pg/tube and was
reproducible from assay to assay. The inter- and intra-assay coefficients of
variability were <15%. The amount of metabolite measured was corrected for
recovery and expressed as pg per ml of blood.
Half-Life of PAF in Human Serum
The half-life of authentic PAF in human serum as a function of PAF-AH activity,
was determined by the method of Stafforini et al (2). Reaction mixtures
containing serum with different PAF-AH activities were incubated with 50 nmol/l
(5 x 10-8 M) [2-acetyl-3H]-PAF (499.5 Bq [2.99 x 10 dpm]/pmol]) at 37oC for
various periods of time. Aliquots (150 µl) were removed at the stated times
directly into an equal volume of 20% acetic acid (for inactivation of PAF-AH in
serum) and the total lipids were then extracted from the samples by the method
of Bligh and Dyer (1). All experiments were repeated in triplicate.
First, the samples were mixed with chloroform /methanol/ water 1:2:0.8 (by vol).
The solvent mixture was phased by adding the appropriate volumes of
chloroform and water to a final ratio of 1:1:0.9. The mixture was shaken
vigorously and the phases were separated by centrifugation. The lower
chloroform phase was removed and evaporated to dryness under a stream of
nitrogen. The residue was dissolved in 50 µl of chloroform and applied to
prepared silica gel G plates. Prior to use, Silica gel G plates were placed in a
tank containing a neutral solvent system of chloroform / methanol / water
(65:35:6, v/v/v). The solvent was allowed to migrate to the top and the plate was
removed and air-dried. It was then activated by heating to 110oC for 30 min. The
activated plates were developed in a solvent system of chloroform / methanol /
water (65/35/6) as previously described (2), using authentic PAF and lyso-PAF
as standards. Lipids were located by iodine staining and the appropriate zone,
co-migrating with PAF standard, was scraped and radioactivity measured in a
liquid scintillation counter.
To evaluate the efficiency of recovery of PAF as well as the reproducibility of the
technique, the same amount of 3[H]-PAF that was added to serum was applied
directly to the thin layer plate without extraction. The efficiency of recovery, 84 ±
8%, was found to be very consistent and satisfactory in 13 independent
Measurement of serum PAF acetylhydrolase activity
The activity of PAF acetylhydrolase was assayed according to the method of
Miwa et al (3) with minor modifications. [2-acetyl-3H]-PAF (1-hexadecyl-2-
[3H]acetyl-sn-glycero-3-phosphocholine and 400 nm of non-radiolabeled PAF
were mixed to a specific radioactivity of 12.5 μCi/μmol. After evaporation of the
solvent under a stream of nitrogen, 1 ml of 0.1 M HEPES buffer (pH 7.2)
containing 1 mM fatty acid-free bovine serum albumin was added, and the
solution was sonicated for 5 min (30 sec increments) at 4°C (Vibra Cell
Sonicator,Sonifier, Model W350, Branson Sonic Corp., Danbury, USA). The
solution was stored frozen for not more than 1 week to avoid non-enzymatic
hydrolysis. The sonication step was repeated each time the substrate was
thawed. The working solutions were 160 μM with 9000-11000 cpm/nmol.
Results were expressed as rate of PAF hydrolysis (amount of released [3H]-
acetate in nmol per ml serum per minute at 37°C). The recovery of released [3H]-
acetate, as estimated by extraction of PAF samples, which had been completely
hydrolysed with 1 M NaOH at 50°C for 30 min, was greater than 96%.
Each assay mixture contained 5 μl of human serum and 10 μl of substrate ([2-
acetyl-3H]-PAF), and the final volume was adjusted to 25 μl with 0.1 M HEPES-
BSA buffer. The final concentration of the substrate was 160 μM. The assay
mixture was incubated for 10 min at 37°C. The reaction was stopped in an ice
bath. The unreacted [2-acetyl-3H]-PAF was immediately bound to an excess of
BSA (16.7 mg/ml final concentration) and precipitated by addition of
trichloroacetic acid (final concentration 8% v/v) for 10 min at 0°C. Denatured
protein was removed by centrifugation at 5000 g for 15 min. The released [3H]
acetate was measured by scintillation counting in 6 ml of ACS scintillation fluid.
Before calculating enzyme activities, blanks were subtracted. Blanks were
prepared using acid treated serum (pH 2.0, 30 min, 37° C) to inactivate PAF
Stability of PAF-AH
Serum samples from individuals with fatal peanut anaphylaxis and from various
control groups may have been stored under differing conditions, subjecting PAF-
AH to thermal stresses. In order to evaluate the effect of handling and storage
conditions on PAF-AH activity, sera were stored at different temperatures. The
stability of PAF-AH stored at 40C and 230C and subjected to repeated cycles of
freeze- thawing was assessed. PAF-AH levels were measured at specified times
following storage at 230C ( 2, 4, 8, 24 and 48 hours) and at 40C (5 and 10 days)
and following 5 sequential cycles of freezing and thawing. To assess the stability
of PAF-AH to long term storage, serum samples stored at -700C since 1998 were
re-assayed for PAF-AH and compared to results of assay results prior to storage.
Stability of PAF-AH: To assess the stability of PAF-AH to long term storage at
-700C, PAF-AH activity was measured in sera (n=25) in 1998 and again in 2006.
Mean PAF-AH activity in fresh sera in 1998 was 27.04 ± 9.92 vs 22.98 ± 7.23
nmol/ml/min in the same serum samples eight years later (p=0.1047). PAF-AH
activity was measured in 6 fresh sera subjected to 5 freeze-thaw cycles.
Residual activity ranged from 90 – 105% of that in fresh sera. Sera (n=6) stored
at 40C for 10 days retained 96 – 102% of initial activity, and sera stored at 230C
for up to 48 hours retained 84 – 92% of PAF-AH activity of fresh sera.
Effect of Epinephrine on PAF-AH Activity: The effect of a typical intramuscular
dose of epinephrine on serum PAF-AH activity was assessed. The pre-injection
baseline plasma epinephrine concentration was 339 ± 115 pg/ml. A maximum
plasma epinephrine concentration of 2136 ± 351 pg/ml was achieved by 8±2 min.
After 40 min, epinephrine concentration was in the range of 400-500 pg/ml, until
180 min. Mean PAF-AH activity prior to injection of epinephrine was 25.4
nmol/min/ml. PAF-AH activity did not change significantly over the course of 3
hours. Serial PAF-AH activity ranged from 28.5 ± 6.6 (SD) to 30.8 ± 6.9
nmol/min/ml over the course of 180 min. following epinephrine injection.
Effect of glucocorticosteroids on PAF-AH Activity: Patients with both LTA and
NLTA were treated with glucocorticosteroids, expressed as micrograms of
beclomethasone equivalent. The large standard deviation relative to the mean of
steroid dosage (mean ± SD, 537 ± 466) suggested the variable was skewed or
was not normally distributed; the lognormal distribution had identical mean and
SD of 5.998 ± 0.758 with median identical to the mean (5.991). The dosages of
steroids were compared between the children with LTA and with NLTA with the t-
test. There was no significant difference in steroid dosage between LTA (mean
644 ± 467) and NLTA (mean 473 ± 470), whether untransformed (p=0.40) or log-
transformed (p=0.29). The asthma type explained very little of the variation (R-
square 3.2% and 5.1% respectively).
1. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification.
Can J Biochem Physiol. 1959;37:911-7.
2. Stafforini DM, Elstad MR, McIntyre TM, et al. Human macrophages secrete
platelet-activating factor acetylhydrolase. J Biol Chem. 1990;265:9682-7.
3. Miwa M, Miyake T, Sugatani J, et al. Characterization of serum platelet-
activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum
PAF acetylhydrolase and respiratory symptoms in asthmatic children. J Clin
02468 10 1214
[3H]PAF remaining ( %)
Supplemental Figure 1. Half-life of exogenous PAF as a function of PAF-AH
activity. a. serum with PAF-AH activity of 7.8 nmol/ml/min; b. serum with PAF-
AH activity of 24.8 nmol/ml/min; c. serum with PAF-AH activity of 47.6
Supplementary Table 1b. Analysis of PAF-AH levels by anaphylaxis Download full-text
grade in subset of patients with allergic reactions to foods
Mean ± SD
(% ≤ 20)
0 0 3