Tsen, Lawrence C. MD*; Tarshis, Jordan MD‡; Denson, Donald D. PhD§; Osathanondh, Rapin MD†; Datta, Sanjay MD*; Bader, Angela M. MD*
Departments of *Anesthesia, Perioperative, and Pain Medicine, and †Obstetrics and Gynecology, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts; ‡Department of Anesthesia, Sunnybrook Hospital, Toronto, Ontario, Canada; and §Department of Anesthesiology, Emory University, Atlanta, GA
June 22, 1999.
Address correspondence and reprint requests to Angela Bader, MD, 75 Francis St., Boston, MA 02115.
Pregnancy-related decreases in protein binding may contribute to altered effects of local anesthetics in the parturient. Previous studies have measured protein binding of bupivacaine in term parturients; the current study defines the ratio of bound-to-free bupivacaine throughout gestation at both therapeutic and toxic systemic concentrations of bupivacaine. Venous samples were obtained from 81 women, including 70 parturients, ranging from 7 to 42 wk of gestation and 11 nonpregnant controls. The percent bound bupivacaine at a fixed concentration was determined for each sample at both therapeutic (1 μg/mL) and toxic (5 μg/mL) concentrations using an ultrafiltration technique. Albumin and α-1-glycoprotein levels were also measured. Linear regression analysis showed a significant increase in concentration of free bupivacaine throughout gestation at the 5-μg/mL concentration, corresponding to a decrease demonstrated in both albumin and α-1-glycoprotein levels. A similar correlation was not found at the 1-μg/mL concentration. Although the relative magnitude of these changes is small, the relative change in free drug throughout gestation is large. Protein binding is only one of several mechanisms that may influence the susceptibility to local anesthetic toxicity in the parturient; however, its relative importance remains unclear.
Implications: When venous samples taken from pregnant women were mixed with 5 μg/ml bupivacaine and analyzed, an increase in the free fraction of drug was seen with increasing gestational age, corresponding to decreases in α-1-glycoprotein and albumin.
Asignificant decrease in serum protein binding of local anesthetics has been reported in the term parturient (1,2). There is controversy as to whether there is an increased incidence of toxic reactions to local anesthetics in the pregnant patient (3,4), but a decrease in protein binding may be a relevant factor. Our group has previously published a study defining the pattern of protein binding throughout gestation with therapeutic systemic levels of lidocaine in the parturient (5). A significant progressive decrease in protein binding was found throughout gestation. Lidocaine is a local anesthetic with intermediate protein binding, and our data raised questions regarding the gestational pattern of protein binding to a more highly bound local anesthetic, such as bupivacaine (6). Our previous study used only therapeutic systemic levels of local anesthetic, so the current study was designed to assess the gestational pattern of protein binding to bupivacaine at both therapeutic and toxic systemic levels. In addition, albumin and α-1-glycoprotein levels were measured in the same group.
The study was approved by our hospital’s Committee for the Protection of Human Subjects, and written, informed consent was obtained from all participants. Venous blood samples were obtained from a group of 81 women, including 11 nonpregnant controls and 70 pregnant patients ranging from 7 to 42 wk of gestation. Both pregnant and nonpregnant participants were taking no medications other than vitamins; in particular, none of the nonpregnant patients were taking oral contraceptives.
Venous blood samples were collected in plain glass blood collection tubes, allowed to clot, and then centrifuged to obtain serum. Serum was separated and stored at −20°C until required for protein binding determination. Serum, rather than plasma, was used to avoid any possible effects of in vitro lipolysis previously reported during pregnancy (7,8). After thawing at room temperature, microliter quantities of bupivacaine HCl solution were added to serum samples to obtain concentrations of either 1 μg/mL or 5 μg/mL, corresponding, respectively, to therapeutic and toxic systemic concentrations of bupivacaine (9,10). Samples were then adjusted to physiologic pH, 7.40 ± 0.02 pH units, with appropriate microliter quantities of 0.1 N HCl or 0.1 N NaOH, and gently agitated at room temperature. Ultrafiltrate of serum was obtained from an aliquot of serum using an ultrafiltration technique previously described (11).
Bupivacaine concentrations were determined using a gas chromatographic technique as previously described (12). The limits of detection were 10 ng/mL, and standard curves ranged from 10 ng/mL to 8 μg/mL. The coefficient of variation for the assays was 6% for the serum studies at each concentration; the coefficient was 10%–12% and 2%–5% for the ultrafiltrate of serum studies at the lower and higher concentrations, respectively. Bupivacaine concentrations deviating more than 10% from the desired concentration were not used in data calculation.
Serum albumin and α-1-acid glycoprotein concentrations were determined by the Division of Laboratory Medicine of Emory University Hospital using a Beckman Array Nephelometer (Beckman Coulter, Fullerton, CA) (13) and commercially available kits (14).
Statistical analysis was perfomred using linear regression analysis and analysis of variance with Fisher’s protected least significant difference for intergroup comparisons where appropriate. A P value of < 0.05% was considered to be significant.
Eighty-one participants were enrolled in the study, of whom 11 were not pregnant, 28 were in the first trimester, 21 in the second trimester, and 21 in the third trimester of pregnancy. Only samples within 10% of the expected serum drug concentrations were used for statistical analysis, resulting in a total of 69 in the 1-μg concentration group (8 nonpregnant, 24 first trimester, 18 second trimester, 19 third trimester) and a total of 67 in the 5-μg concentration group (7 nonpregnant, 22 first trimester, 19 second trimester, 19 third trimester).
Based on linear regression analysis of individual samples, the percent bound fraction of bupivacaine decreased significantly throughout gestation at toxic but not at therapeutic serum levels (Figures 1 and 2). Albumin and α-1-glycoprotein levels also decreased significantly throughout gestation (Figures 3 and 4).
Table 1 shows percent of bound bupivacaine and serum concentrations of bupivacaine for the nonpregnant controls and each trimester group. Statistical comparison of the fraction of bound bupivacaine revealed no differences at the 1-μg concentration, whereas a significant difference between the first and third trimester groups was seen at the 5-μg concentrations. Table 2 shows the albumin and α-1-glycoprotein results. Albumin concentrations significantly lessened with each trimester. A decrease in α-1-glycoprotein levels was seen between the nonpregnant controls and the third trimester patients, as well as between first and third trimester groups.
There is controversy as to whether pregnancy is associated with an increased incidence of toxic reactions to local anesthetics. Whereas earlier in vivo (3) and in vitro (15) studies suggested increased toxicity, more recent investigations using the in vivo ewe model have suggested that the toxicity of mepivacaine (16), lidocaine (17), ropivacaine (4), and bupivacaine (4) is not increased during pregnancy. Differences in individual responses to local anesthetics may be the result of alterations in nerve sensitivity, which have been documented as early as the first trimester (18).
Our group previously published a study defining the pattern of protein binding throughout gestation with therapeutic serum levels of lidocaine (2 μg/mL) in the parturient (5). Increasing gestational age was shown to correspond with a decrease in protein binding. Our findings prompted further inquiry in the following two areas: the alterations in protein binding at different concentrations of local anesthetics; and the changes in protein binding experienced with local anesthetic concentrations, which are associated with systemic toxicity. Moreover, we felt it necessary to investigate the protein binding alterations for the local anesthetic most commonly used in the obstetric population, i.e., bupivacaine. Important differences in pharmacokinetics could affect the interaction between local anesthetics and serum proteins: lidocaine has an intermediate degree of protein binding, and a high hepatic extraction ratio; bupivacaine, in contrast, is a drug with a high degree of protein binding, and a low hepatic extraction ratio (19).
For these reasons, we felt that, to better understand the pattern of protein binding to local anesthetics during gestation, data regarding bupivacaine at both therapeutic and toxic plasma levels should be reported. Our data demonstrate that, at the lower therapeutic serum level of bupivacaine, there was no significant correlation between the percentage of bound drug and gestational age. In contrast, at toxic serum levels, a significant decrease in the percentage of bound bupivacaine is seen with increasing gestational age, corresponding to an overall decrease in protein binding. Although the reasons for this are not completely clear, we can speculate that this increase in unbound bupivacaine at toxic serum levels reflects the binding capacities of two proteins: α-1-acid glycoprotein, a high affinity, low capacity site; and albumin, a low affinity, high capacity site. As previously discussed (11), at therapeutic levels of bupivacaine, the high affinity α-1-glycoprotein sites may only be partially saturated; in contrast, at toxic concentrations of bupivacaine, they may be completely saturated. Because the low affinity albumin sites may only be able to bind a certain amount of bupivacaine, regardless of the concentration of bupivacaine, these may be an overall higher unbound fraction of bupivacaine at higher concentrations.
Increasing progesterone levels has not been shown to displace bupivacaine from serum protein binding sites (20). Both progesterone and bupivacaine bind to α-1-acid glycoprotein with similar affinity, but it was apparent that different sites are involved (20). Therefore, the decrease in protein binding throughout gestation is more likely a result of the hormonally induced decreases in concentration of α-1-acid glycoprotein known to exist in the term parturient (2,21) and similarly demonstrated in our study. In term parturients, the number of bupivacaine protein binding sites was shown to be half that of nonpregnant control patients, supporting the differences in concentration of α-1-acid glycoprotein (2,21).
As gestation progresses, the same serum bupivacaine concentration is associated with an increasing free (unbound) concentration. This, in turn, is reflected by an increasing concentration gradient of free drug available for placental transfer and uptake, as well as for maternal vital organ uptake. The effects of protein binding on the susceptibility to bupivacaine cardiotoxicity associated with pregnancy are unclear; earlier work in animals suggested an increased susceptibility (3) that has not been shown in animals for either lidocaine or mepivacaine (17). More recent work, however, does not show enhanced cardiotoxicity of bupivacine (4). Other mechanisms of systemic toxicity to local anesthetics in the parturient may include anesthetic-sodium channel interactions (22), hypoglycemic states, hypomagnesemic states, relative plasma cholinesterase deficiencies, and other changes created by alterations in the physiologic state.
In summary, a decrease in the bound fraction of bupivacaine was demonstrated at higher serum levels with increasing gestational age. Protein binding is only one of several mechanisms that may influence the individual susceptibility to local anesthetic toxicity; however, its relative importance remains unclear.
1. Denson DD, Coyle DE, Thompson GA, et al. Bupivacaine protein binding in the term parturient: : effects of lactic acidosis. Clin Pharmacol Ther 1985; 35: 702–9.
2. Wulf H, Munstedt P, Maier C. Plasma protein binding of bupivacaine in pregnant women at term. Acta Anaesthesiol Scand 1991; 35: 129–33.
3. Feldman HS, Arthur GR, Covino BG. Comparative systemic toxicity of convulsant and supraconvulsant doses of intravenous ropivacaine, bupivacaine, and lidocaine in the conscious dog. Anesth Analg 1989; 69: 794–801.
4. Santos AC, Arthur GR, Wlody D, et al. Comparative systemic toxicity of ropivacaine and bupivacaine in nonpregnant and pregnant ewes. Anesthesiology 1995; 82: 734–40.
5. Fragneto RY, Bader AM, Rosinia F, et al. Measurements of protein binding throughout pregnancy. Anesth Analg 1994; 79: 295–7.
6. Lin SK. The effect of pregnancy on the plasma protein binding of lidocaine: : does it matter? [letter]. Anesth Analg 1995; 80: 1063–4.
7. Ridd MJ, Brown KF, Moore RG, Nation RL. Drug plasma binding and non-esterified fatty acids: : methodologic considerations. Int J Pharm 1982; 11: 11–20.
8. Brown JE, Kitchell BB, Bjornsson TD, Shand DG. The artifactual nature of heparin induced drug protein binding alterations. Clin Pharmacol Ther 1981; 30: 636–43.
9. Datta S, Alper MH, Ostheimer GW, et al. Effects of maternal position on epidural anesthesia for cesarean section, acid-base status, and bupivacaine concentrations at delivery. Anesthesiology 1979; 50: 205–9.
10. Muson ES, Tucker WK, Ausinsch G, et al. Etidocaine, bupivacaine and lidocaine seizure thresholds in monkeys. Anesthesiology 1975; 42: 471–8.
11. Denson DD, Coyle DE, Thompson GA, Myers JA. The role of alpha-1-acid glycoprotein and albumin in human serum binding of bupivacaine. Clin Pharmacol Ther 1984; 35: 409–15.
12. Coyle DE, Denson DD. Simultaneous measurement of bupivacaine, etidocaine, lidocaine, meperidine and methadone. Ther Drug Monit 1986; 8: 98–101.
13. Davis ML, Austin C, Messmer BL, et al. IFCC-standardized pediatric reference intervals for 10 serum proteins using the Beckman Array 360 system. Clin Biochem 1996; 29: 489–92.
14. Salkie ML. A retrospective study of the relative utility of electrophoresis, immunoelectrophoresis, immunofixation, and nephelometry in the investigation of serum proteins. Clin Biochem 1996; 29: 39–42.
15. Datta S, Lambert DH, Gregus J, et al. Differential sensitivities of mammalian nerve fibers during pregnancy. Anesth Analg 1983; 62: 1070–2.
16. Santos AC, Pedersen H, Harmon TW, et al. Does pregnancy alter the systemic toxicity of local anesthetics? Anesthesiology 1989; 70: 991–5.
17. Morishima HO, Finster M, Arthur GR, Covino BG. Pregnancy does not alter lidocaine toxicity. Am J Obstet Gynecol 1990; 162: 1320–4.
18. Butterworth JF, Walker FO, Lysak SZ. Pregnancy increases median nerve susceptibility to lidocaine. Anesthesiology 1990; 72: 962–5.
19. Arthur GR. Pharmacokinetics of local anesthetics. Handbook Exp Pharmacol 1987; 81: 165–86.
20. Coyle DE, Denson DD, Essell SK, Santos DJ. The effect of non-esterified fatty acids and progesterone on bupivacaine protein binding. Clin Pharmacol Ther 1986; 39: 559–63.
21. Song CS, Merkatz IR, Rifkind AB, et al. The influence of pregnancy and oral contraceptive steroids on the concentration of plasma proteins. Am J Obstet Gynecol 1970; 108: 227–31.
22. Clarkson CW, Hondeghem LM. Mechanism for bupivacaine depression of cardiac conduction. Anesthesiology 1985; 62: 396–405.