Alpha‐1‐acid glycoprotein and its potential impact on local anesthetic dose in neonates

Alpha‐1‐acid glycoprotein is an acute‐phase protein with a high affinity for amide local anesthetics. Compared to adults, neonates have lower concentrations of this glycoprotein in plasma, and are therefore at higher risk of developing local anesthetic toxicity. Alpha‐1‐acid glycoprotein concentrations rise in adults after surgery as a response to stress as well as in inflammatory conditions. Previous studies have shown that concentrations of alpha‐1‐acid‐glycoprotein in neonates vary postpartum, influenced by gestational age and mode of delivery.


| INTRODUC TI ON
Contrary to what was previously believed, neonates do experience and remember pain. 1,2 Therefore, neonates subjected to painful procedures need effective and safe pain management. Systemic opioids are often the drug of choice, but are associated with adverse effects, such as respiratory depression, obstipation, and may even have negative effects of the developing brain. 3,4 Results from recent studies and clinical experience has led to an increased use of local anesthetic methods in this age group. 5,6 Methods, which are widely used in adults and older children with good results. 7 However, local anesthetics (LA) may have serious adverse effects such as seizures and hemodynamic instability, 8,9 due to high dosage or unintentional intravascular administration.
The effect and toxicity of LA are both exercised by its unbound fraction, which subsequently is dependent on binding to plasma proteins. Alpha-1-acid glycoprotein (AAGP) is an acutephase protein with high affinity to amide LA. 10 It is known that neonates have low concentrations of AAGP compared to adults. 11,16 In adults, as well as in children, AAGP plasma concentrations rise as a response to stress, not only in patients undergoing major surgery, but also at some inflammatory conditions. [13][14][15][16] It has been shown that AAGP concentrations in newborns increases with gestational age, and are also influenced by mode of delivery, with higher values related to vaginal birth. 12 A dose reduction of LA is recommended for neonates to avoid potential local anesthesia systemic toxicity (LAST), which partly might be due to the expected lower concentrations of AAGP. However, LAST is a very rare adverse effect in neonates, when recommended doses are administrated. 12,[17][18][19][20] The primary aim of this prospective observational study was to compare the plasma concentrations of AAGP pre-and postoperatively in neonates undergoing major surgery. Secondary aims were to determine any correlation between AAGP concentrations and sex, birthweight, mode of delivery, C-reactive protein (CRP) concentrations or gestational age.
The study was performed in accordance with the Declaration of Helsinki and is registered in the Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12622000159752. After written parental informed consent, 25 neonates (<3 months of age), scheduled for major surgery and with a need of postoperative intensive care, were included in the study. Inclusion was performed between May 2019 and October 2020. Exclusion criteria were the need of extracorporeal membrane oxygenation (ECMO), suspected severe genetical disorder or denied parental consent.
All the patients received general anesthesia according to clinical routine. Anesthesia was induced by fentanyl 1-2 μg kg −1 , propofol 2-6 mg kg −1 , or thiopental 5-7 mg kg −1 after which endotracheal intubation was facilitated by rocuronium 1 mg kg −1 or atracurium 0.5 mg kg −1 . Anesthesia was maintained by sevoflurane (2-4% in an air/oxygen mixture) and complemented with opioids (fentanyl or morphine) as needed. A total of eight patients received LA continuously with levobupivacaine during and after surgery and three patients received caudal single dose with levobupivacaine, as presented in Table 1.

| Blood sampling and measurements
Venous blood samples (0.5 mL) were drawn into EDTA-tubes

What is already known
• Low alpha-1-acid glycoprotein concentrations are associated with reduced binding of amide local anesthetics which may interfere with toxicity and effect of the drugs.
• Adults and older children have the ability to respond to surgery with elevated concentrations of alpha-1-acid glycoprotein.

What this article adds
• Concentrations of alpha-1-acid glycoprotein are higher postoperatively than preoperatively, in neonates.
• Our finding indicates that the risk of developing local anesthetic toxicity due to low concentrations of alpha-1-acid glycoprotein is not increased in neonates.
• Our data on neonates adds important knowledge and complements former studies concerning alpha-1-acid glycoprotein concentrations in older children and adults.
Both assays were analyzed on the Corbas C system: (Roche Diagnostics). Endogenous interferents, hemolysis, icterus, and lipemia were measured and was below specified criteria for all samples.

| Statistical analyses
Data are reported as number of observations (n) and median values (inter-quartile range (IQR)) unless otherwise stated. Two independent groups of samples were compared using the Mann-Whitney U test. Kruskal-Wallis test for the comparison of several independent observations. Confidence intervals for single proportions was evaluated using exact binomial tail areas. Correlations between AAGP con-

| RE SULTS
Patient characteristics are presented in Tables 1 and 2. All patients were possible to include in the statistical analyses. 78 % of the intended samples were collected. Omitted sample collections were due to lack of indwelling catheters, protocol violation, and limitations due to small blood volumes. Three samples could not be analyzed at the laboratory due to inadequate sample volume after processing.
Data are available in Table S1.

| Plasma concentrations of AAGP in relation to CRP
Concentration of CRP was measured in all patients according to clinical routine. A correlation was determined between CRP and AAGP, both rising postoperatively in a similar manner, r s = 0.5976; p < 0.001.
These data are presented in Figure 2

| Plasma concentrations of AAGP in relation to postnatal age
AAGP concentrations in neonates (measured from venous or arterial lines) at 3 days of age (median), were higher than directly postpartum (measured from cord blood), in our previous study, 0.300 g L −1 as compared to 0.158 g L −1 (p < 0.001). 12 Data from both studies are presented in Figure 3.
It was not possible to demonstrate any influence of sex on AAGP concentrations in the present study. Neither was it possible to determine any correlation between AAGP and postnatal age, gesta-

| DISCUSS ION
The main finding of the present study is that AAGP plasma concentrations in neonates increases after surgery with higher values at 24 and 48 h after completed surgery. This finding is important when determining safe and effective dosing of amide LA in neonates.
Postoperative pain in neonates is still commonly managed with opioids despite their many well-known adverse effects. 4 An alternative, or complement, to opioids are regional anesthesia (RA) for example wound catheter, epidural, or peripheral nerve blocks. The current recommendations of reduced dosage of LA to neonates, compared to older children, are partly based on proven experience as well as studies made 25 years ago. 21 Many of the studies conducted in this age group have very few patients included for obvious reasons. We therefore believe that results from the present study can be of value, complementing previous findings of others.
Our results could also be included in population pharmacokinetic modeling studies such as conducted by Aarons and Vashisht and coworkers. 15,16 Our data are unique with a homogenous group of very young patients. Data are available in Table S1.
To reduce the risk of LAST in neonates and infants, Booker and co-workers 19  Booker and co-workers, showed a sex difference in preoperative AAGP concentrations (higher concentrations in male infants), a difference not possible to replicate in the present study, possibly due to our small study population. 19 In the two largest diagnostic groups, congenital diaphragm hernia and esophageal atresia, there was no statistical difference regarding the rise of AAGP concentrations in plasma. This indicates that from a safety aspect the same dose regime might be used in different type of major surgery.
AAGP is an acute phase protein, produced in the liver, this also applies for CRP which is a marker for inflammatory response. 22 Earlier studies have shown that the concentration of AAGP in infected patients were higher, compared to those in healthy patients. 13,23 In the present study all patients were included into the F I G U R E 2 Correlation between alpha-1-acid glycoprotein (AAGP) concentrations in newborn infants and C-reactive protein (CRP).

F I G U R E 3
Median alpha-1-acid glycoprotein (AAGP) concentrations in newborn infants at delivery and at 3 days of age.
analysis, including those with an upgoing trend regarding CRP. It was possible to determine a correlation between AAGP and CRP.
This implies that it might be possible to use the AAGP concentration as a marker for inflammation and even for infection. 13,24 Patients with elevated CRP will most likely have higher concentrations of AAGP, indicating that they might tolerate higher doses of LA, if needed.
Ten patients received fresh frozen plasma from adult donors during or after surgery due to clinical reasons. Theoretically, it may be conceivable that those patients had higher AAGP because of this added plasma. However, due to the limited number of subjects it was not possible to determine a statistically significant difference compared to the rest of the study population.
When determining adequate dosing of LA in neonates, the most adopted regime is to reduce the dose of bolus and maintenance infusion with 40%, respectively 50% compared to older children. 21,25 The reason for dose reduction is based on the fact that it is the unbound fraction of amide LA that is important for both the effect and toxicity. Neonates have immature liver function and a reduced capacity for producing glycoproteins, that could lead to higher risk of LAST. A risk that further increases during infusion of LA due to reduced clearance in neonates. Incomplete myelination at birth is another reason why lower dose of LA is sufficient in neonates.
Neonates cannot display early signs of LAST, such as discomfort and mild neurological signs, which must be taken under consideration. Thus, it is obvious that we must have a higher safety margin in this age group to avoid LAST, but it is also important that we offer sufficient pain relief. Previous studies have shown that extremely preterm infants have very low plasma concentrations of LA after infusion with levobupivacaine (with existing dose recommendations) and no signs of LAST were seen. 17,18 The result in the present study implies that neonates can respond to surgical stress with a significant rise in AAGP and therefore, theoretically reducing the risk of LAST. Thus, based on present and earlier studies by our group and others, 12,15,16,19 we recommend that slightly higher doses of LA may be reasonable to consider if needed for pain control. In an ongoing randomized study, we aim to measure concentrations of AAGP and LA in plasma in children up to 3 months of age undergoing major surgery, in order to further optimize dose recommendations in this age group.

| Study limitations
When including small children in studies, there is a limit to the amount of blood that can be sampled, both in terms of sample volume and in numbers of blood samples. Since no extra punctures were acceptable (according to the ethical approval), some of the samples were impossible to accomplish, leading to missing values.
Three out of the 97 blood samples collected in the study, were not possible to analyze due to inadequate sample volume after processing. None of the samples were below the lower limit of quantification for the instrument used.

| CON CLUS ION
The primary finding of our study is that AAGP concentrations increase significantly in neonates as a response to surgical stress.
Secondarily, gestational age, sex, or mode of delivery did not have any influence on plasma concentration of AAGP in this study, possibly due to small study population. A correlation between AAGP and CRP was found with simultaneously rising values.

ACK N OWLED G M ENTS
To all participating children and their parents for giving their consent. All nurses at the wards and the research nurses, helping with the blood sampling.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.