Alfaxan® (alfaxalone) is a steroid general anesthetic widely used in veterinary medicine for induction and maintenance of anesthesia in several species. While the use of alfaxalone in veterinary practice has several benefits compared to the use of other anesthetic agents, the fact that it is derived from progesterone may confound the measurement of the latter in the blood of animals under alfaxalone treatment. In the present case study, we report the measurement of serum progesterone in an individual common marmoset (Callithrix jacchus) during five ovarian cycles in which luteolysis was
induced by PGF2α. Blood samples were usually taken from the awake
animal with the exception of the fifth cycle in which the sample was
collected under alfaxalone anesthesia in connection with a tooth extraction. In contrast to the previous four cycles in which luteolysis resulted in the expected marked decrease in progesterone concentrations, the – apparent – progesterone level in the cycle under alfaxalone treatment remained unexpectedly high. Cross-reactivity of the non-specific antibody used in the progesterone assay with alfaxalone most likely explains this finding.
Introduction
Alfaxalone is a veterinary general anesthetic which enhances the inhibitory
effects of γ-aminobutyric acid (GABA) on GABAA receptors,
causing nerve cell hyperpolarization and blocking neural impulse
transmission (Ghit et al., 2021; Papich, 2016; Albertson et al., 1992).
It shows certain benefits compared to other anesthetic agents due to a broad
margin of safety and a low cumulative effect after repeated doses, which
enables a rapid recovery (Lau et al., 2013; Ferre et al., 2006; Rezende,
2015). Alfaxalone is used for both induction and maintenance of anesthesia
in many species including companion and experimental animal species such as
dog (White and Yates, 2017), cat (Khenissi et al., 2017), goat
(Abouelfetouh et al., 2021), sheep (Andaluz et al., 2012), pig (Bigby et al.,
2017), horse (Goodwin et al., 2011), rhesus
macaque (Bertrand et al., 2017), cynomolgus monkey (Casoni et al.,
2015), and common marmoset (Bakker et al., 2013). Alfaxalone anesthesia
has minimal negative impact on the cardiorespiratory system though it can
cause hypoxemia if administrated in high doses (Wada et al., 2020). It is
also commonly used without side effects in pregnant dogs showing no negative
effects on the stability of maternal and fetal hemodynamics (Andaluz et
al., 2013; Metcalfe et al., 2014). Altogether, alfaxalone is regarded as a
safe anesthetic agent and is widely used in high-risk patients (Bosing et
al., 2012). However, it has a very poor analgesic effect (Bennell et al.,
2019).
Chemically, alfaxalone is a synthetic pregnane steroid, namely 3α-hydroxy-5α-pregnan-11,20-dion (Fig. 1). It is a derivative of
progesterone and allopregnanolone, differing from the latter only by the
addition of a ketone group at C11, though it has no glucocorticoid or
mineralocorticoid action (Ferre et al., 2006; Rezende, 2015).
Chemical structure of progesterone and alfaxalone.
While the use of alfaxalone in veterinary practice has several advantages
compared to the use of other anesthetic agents as mentioned above, the fact
that it is derived from progesterone and is structurally similar to
naturally occurring pregnanediones and pregnanolones suggests that it may
confound the measurement of progesterone in the blood of animals under
alfaxalone treatment. This is due to the fact that antibodies used for the
quantification of progesterone by immunological methods (e.g., ELISA, RIA)
often cross-react with 5α- or 5ß-reduced progestogens
(Graham et al., 2001) to which alfaxalone belongs. A co-measurement
of alfaxalone in serum progesterone assays might therefore be possible (as
also suggested by findings in cats; Trumble et al., 2020), even
if the degree of cross-reactivity with the progesterone antibody may be low.
Here, we report a case of an unusually high serum progesterone value in a female common marmoset anesthetized by intramuscular injection of alfaxalone
during the follicular phase of its ovarian cycle.
Common marmosets (Callithrix jacchus) are widely used non-human primates in biomedical research and translational medicine (Kishi et al., 2014). Studies conducted in these areas often require a detailed monitoring of female reproductive condition, including information on ovarian activity and the exact state of the ovarian cycle (Drummer et al., 2021). Such
profound knowledge of the reproductive cycle is required for instance to
monitor hormonal stimulation or to determine the optimal time points for
oocyte or embryo retrieval and embryo transfer (Drummer et al., 2021;
Marshall et al., 2003). Determining the female reproductive status,
including ovarian cycle stage, can be achieved through measurements of
progesterone in small blood samples which are usually collected from awake
animals (Saltzman et al., 1997). Based on a
threshold value of 10 ng mL-1 progesterone indicating occurrence of ovulation
in the marmoset, the follicular and luteal stages of the ovarian cycle can
be reliably determined (Harlow et al., 1983). Thus, any
confound in the progesterone measurement could lead to a false
interpretation of a female's cycle stage, and in this
respect, caution might be advised for serum progesterone determination in
samples collected under alfaxalone treatment (Trumble et al.,
2020). Here we describe a case study of an unexpectedly elevated
progesterone value with the blood sample taken under alfaxalone anesthesia
in a female common marmoset. Since alfaxalone is structurally very similar
to allopregnanolone (see above), which shows a 64 % cross-reactivity in our progesterone assay (Graham et al., 2001), we assessed the
probability that interference of alfaxalone in our progesterone assay due to
potential cross-reactivity was responsible for the unexpectedly high progesterone value recorded.
Materials and methodsAnimal
We describe the case of a 4-year-old female common marmoset (animal number
no. 17402) enrolled as an oocyte donor in an experiment aiming at the
generation of genetically modified animals. The animal was obtained from the
breeding colony of the German Primate Center and the experimental procedures
were authorized by the federal authorities (LAVES) under license number
33.19-42502-04-19/3221. Health and general condition of the animal were
controlled daily by experienced animal care takers and regularly by
veterinarians. The animal was pair-housed with an intact male and was fed
a marmoset-specific diet, supplemented with fruits and vegetables as described
elsewhere (Drummer et al., 2021). Water was available ad libitum.
Ovarian stimulation and blood sampling protocol in
the reported case. Routinely, blood collection is performed on day 2 on the
non-anesthetized animal.
The animal's ovarian cycle was routinely monitored twice a
week by blood collection from the femoral vein without anesthesia in an
awake condition, to determine progesterone concentration. In the mid-luteal
phase synthetic prostaglandin F2α (PGF2α; 0.2 mL of a
mixture of 0.1 mL Estrumate®
250 µg mL-1,
Intervet Deutschland GmbH, Unterschleißheim, Germany, and 3.2 mL Ringer
lactate solution, B. Braun SE, Melsungen, Germany) was injected into the
femoral musculature in order to induce luteolysis and thus to initiate a new
follicular phase (Summers et al., 1985). The day
after PGF2α administration and the 8 following days, the animal was
given 25 IU FSH (follicle-stimulating hormone, GONAL-f® 450 IU/0.75 mL, Merck Europe B.V., Amsterdam, the Netherlands) intramuscularly
(femoral musculature) to stimulate the ovaries. On the second day of FSH
application, we detected a brownish, broken canine in the right upper jaw
and decided to remove the tooth. The animal was anesthetized with a
combination of diazepam (0.05 mL per animal,
Diazepam-ratiopharm® 10 mg/2 mL, Ratiopharm GmbH, Ulm, Germany) and alfaxalone (0.1 mL per 100 g body weight, 0.4 mL in total, Alfaxan® 10 mg mL-1, Jurox, Dublin, Ireland), blood for routine
progesterone measurement was collected under anesthesia, and the canine was
removed afterwards. Appropriate analgesics and antibiotics were applied. The
animal recovered from anesthesia without any complications. Three and 7
days later (i.e., on days 5 and 9 post PGF2α administration)
additional blood samples were collected for progesterone monitoring with the
animal being awake and non-sedated (Fig. 2).
Progesterone measurement and alfaxalone cross-reactivity
Progesterone was determined in the blood serum by a direct, non-extraction
enzyme immunoassay (EIA) using a monoclonal antibody (Quidel clone no. 425;
CL425) produced against 11-hydroxyprogesterone-hemisuccinate: BSA
(Grieger et al., 1990), and progesterone–horseradish
peroxidase (HRP) was used as a conjugate. Peripheral plasma progesterone
measurement is routinely used for ovarian cycle monitoring in our female
common marmosets (Harlow et al., 1983). The assay has been
analytically validated by demonstrating (i) high sensitivity (90 % binding = 50 pg mL-1), (ii) parallelism between displacement curves from standards and dilutions of plasma, and (iii) precision (intra- and interassay CVs of high and low value quality controls =< 10 % and < 15 %, respectively). Moreover, the assay produces biologically valid results by demonstrating the reliable discrimination of ovarian cycle stages in the marmoset, with progesterone concentrations < 10 and
> 10 ng mL-1 indicating the follicular and luteal phase of the
ovarian cycle, respectively (Harlow et al., 1983) (see Fig. 3
as example).
Cross-reactivity determination of alfaxalone
measurement in the progesterone enzyme immunoassay used for analysis of
serum progesterone concentrations of common marmosets. Concentration values
at 50 % binding (50 % B0) for progesterone and alfaxalone used for
calculation of the alfaxalone cross-reactivity data are indicated.
The assay's antibody has a high cross-reactivity with
progesterone but also substantially cross-reacts with other pregnanediones
as well as with pregnanolones (Graham et al., 2001). Due to its
non-specificity, the EIA has been widely used for monitoring progesterone
and progesterone metabolites in blood and feces, respectively, of a variety
of species (Graham et al., 2001).
Since we assumed that alfaxalone would interfere in the measurement of serum
progesterone (see Introduction), we determined its degree of
cross-reactivity in our progesterone assay and found it to be 4.4 %
(Fig. 3).
Results
Under normal conditions (i.e., when blood was collected in a non-sedated
state), the animal reacted to the application of the PGF2α with the
expected decline of progesterone concentrations to ≤ 10 ng mL-1 within 1–2 d, indicative of the onset of a new follicular phase (Fig. 4, circles) (Summers et al., 1985). In the sample collected under
alfaxalone anesthesia 2 d after induction of luteolysis, however, the
determined (apparent) serum progesterone concentration was not decreased but
stayed elevated (Fig. 4, hexagon), suggesting that the animal might not
have responded to the PGF2α administration. However, in the samples
collected on days 5 and 9 after PGF2α injection progesterone
concentrations were < 10 ng mL-1 (Fig. 4, rectangle), indicating a
successful luteolysis and showing that the animal had indeed responded
properly to the PGF2α treatment like in previous cycles. The
markedly elevated progesterone value on day 2 post PGF2α treatment
was therefore most likely related to the alfaxalone anesthesia, which we
discuss in detail in the following.
Serum progesterone levels in the animal #17402
after the intramuscular injections of PGF2α (PGF) during several
ovarian cycles. The sample highlighted by the hexagon was taken under
alfaxalone anesthesia. Note that this sample was taken 2 d after
PGF2α application, which caused the expected marked decline in
progesterone concentrations in the previous cycles.
Discussion
Alfaxan® (alfaxalone) is an anesthetic agent widely used in veterinary medicine for induction and immersion of anesthesia (Rezende, 2015). We frequently use alfaxalone in marmosets for short-term anesthesia as it is well tolerated, safe and the animals wake up fast and gently (Bakker et al., 2013). The common marmoset (Callithrix jacchus) is a useful animal model in
translational medicine (Kishi et al., 2014). One big
advantage of this model is the small size of the animals, which facilitates
handling, including blood sample collection for hormone measurements and
other blood chemistry diagnostics, without anesthesia
(Saltzman et al., 1997). The case we describe
here refers to an animal enrolled in an experiment for the generation of
transgenic marmosets. For this purpose, we monitored the ovarian cycle by
measuring the blood progesterone levels twice a week. In contrast to
previous cycles in which blood was collected without anesthesia, we found an
unexpectedly elevated progesterone value following luteolysis in the sample
collected under anesthesia. We speculated that alfaxalone might have
confounded the measurement of native progesterone in this sample,
particularly because our progesterone antibody substantially cross-reacts
with 5α-reduced pregnanediones and pregnanolones (Graham et
al., 2001) to which, structurally, alfaxalone belongs. We determined a
cross-reactivity for alfaxalone of 4.4 % in our progesterone EIA, a
relatively low value. However, because the dosage of alfaxalone required for
sedation is rather high (1 mg per 100 g body weight; 4 mg in total for the study animal), we calculated that even if only 20 % of the injected alfaxalone was present in the circulation during the time when the blood sample was collected, our progesterone immunoreactivity value would have been raised by about 30 ng mL-1. Thus, co-measurement of alfaxalone in our progesterone EIA
was substantial and significantly interfered with the immunological
determination of blood progesterone levels (Trumble et al., 2020).
This was indirectly confirmed by the finding that in the samples collected
on days 5 and 9 after PGF2α injection when the animal was not
sedated, the progesterone concentrations showed the expected low values,
indicating that the animal had indeed responded properly to the PGF2α treatment like in previous cycles. Since alfaxalone, despite being a
progesterone derivative, does not bind to glucocorticoid, mineralocorticoid
or sex hormone receptors (Ferre et al., 2006), we
assume that the observed interference was not due to an increased alfaxalone-induced progesterone production.
Given that pregnanediones and pregnanolones are abundant metabolites of
progesterone in the urine and feces of many animal species (Graham
et al., 2001) and considering that alfaxalone metabolism takes place mainly
in the liver but also in the kidney and lung
(Ferre et al., 2006), we envisage that the
measurement of urinary/fecal progestogens in non-invasive samples collected
from animals which had received alfaxalone anaesthesia may also be
confounded by excreted alfaxalone metabolites. This needs to be confirmed
though.
Generally, the use of a highly specific progesterone antibody should
substantially reduce or even overcome the problem of co-measurement of
alfaxalone. To estimate the potential risk of a confounding co-measurement
as encountered by us, we recommend to determine the alfaxalone
cross-reactivity in the progesterone assay of choice prior to the collection
of blood samples under alfaxalone anesthesia.
As alfaxalone does not interact with the hormonal steroid receptors
(Visser et al., 2002) we do not expect any
progesterone-like biological function of the anesthetic agent and therefore
no unfavorable impact on the female reproductive system. Even if there is a
biological effect of alfaxalone, this would be presumably only short-term
given that the half-life of the compound is relatively short. Mean terminal
plasma half-life in the recommended clinical dose is about 24 min in
dogs (Ferre et al., 2006) and 45 min in cats (Whittem et al., 2008) after a single i.v. application of 2 and 5 mg kg-1 respectively.
Progesterone measurement in blood serum is a routine analysis in veterinary
reproduction medicine (Brugger et al., 2011; Saltzman et al., 1997;
Hammer and Howland, 1991). Mostly, blood collection procedures are well
tolerated by animals, at least domesticated and/or small-bodied ones and
those habituated to the procedure (Saltzman et
al., 1997). In larger-bodied non-domesticated species and in cases of
uncooperative or aggressive animals, sedation is required prior to the blood-taking procedure (Hotchkiss and Young, 2020). Our present
finding shows that in such cases attention should be paid on the selection
of the anesthetic agent when the blood sampling is also undertaken for
progestogen measurements. Under such conditions the use of alfaxalone as the
anesthetic agent should be avoided as it may confound the progesterone
determination. If unnoticed, this may lead to false interpretations
regarding progesterone values with potentially far-reaching consequences,
such as break-off of artificial reproduction technologies, like ovarian
stimulation treatments in animals selected for oocyte donor studies. In the case
of a concomitant veterinary intervention in anesthesia, it is preferable to
do all the procedures in the anesthetized animal as this minimizes the
stress for the monkeys and is consistent with animal welfare. If
progesterone determination is required from animals under anesthesia, the
combined application of xylazine and ketamine or medetomidine and ketamine
could be an adequate alternative to alfaxalone (Goodroe
et al., 2021).
We conclude that alfaxalone can confound immunoassay-based progesterone
determination and should be avoided for anesthesia prior to blood sampling,
when the progesterone blood level has to be determined.
Data availability
All raw data can be provided by the corresponding authors upon request.
Author contributions
MD and MH conceived the idea. MD and MH analyzed the data. MD, MH, CD, RB designed and performed the experiment. MD and MH wrote the manuscript. CD and RB reviewed and edited the manuscript. All authors discussed the results and commented on the manuscript.
Competing interests
The contact author has declared that none of the authors has any competing interests.
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Financial support
The publication of this article was funded by the Open Access Fund of the Leibniz Association.
Review statement
This paper was edited by Eberhard Fuchs and reviewed by three anonymous referees.
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