JPM Vol. 28, No. 3 November 1992:159-166
Determination of Acetazolamide in Human Serum by Enzymatic Assay Ingrid M. G r e e n e and A l e x a n d e r D. K e n n y Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A.
Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), formerly used as diuretics, still play a role in the treatment of glaucoma, epilepsy, and altitude sickness. There is now hard evidence from both in vitro and in vivo studies in animals that carbonic anhydrase plays a vital function in bone loss. Acetazolamide blocks bone resorption in these experimental models. We have postulated that acetazolamide has potential for the treatment of human conditions associated with bone loss. In preparation for a clinical trial of acetazolamide's effectiveness in this regard, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum. Acetazolamide is stripped from binding to serum proteins by adding 10-6 M salicylic acid and adjusting the pH to 2.5, followed by ultrafiltration through a membrane (10 kD cutoff). The latter permits the free acetazolamide to enter the filtrate but retains any carbonic anhydrase (31 kD) which may contaminate the serum from hemolysis. The carbonic anhydrase inhibitory activity in the filtrate, representing the acetazolamide, is determined in a carbonic anhydrase assay using acetazolamide as the standard. Recoveries of acetazolamide added to human serum ranged from 83% to 94% depending on the concentration. Precision, as judged by the coefficient of variation, was 10.5%.
Keywords: Acetazolamide; Human serum; Method
Introduction Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), were formerly used as diuretics (Maren, 1984) until supplanted by more effective agents. Nevertheless, they still play a role in the treatment of glaucoma, epilepsy, and altitude sickness (Wistrand, 1984). Reports since 1970 have indicated that the CA inhibitor, acetazolamide, has important in vivo and in vitro effects on bone and calcium metabolism. In vivo, acetazolamide inhibits the hypercalcemic responses to parathyroid hormone (PTH) and dibutyryl-cAMP (DBcAMP) (Waite and Kenny, 1970; Waite et al., 1970; Waite, 1972) and partially inhibits disuse osteoporosis in a rat model (Conaway et al., 1973; Kenny, 1985a, b) at doses which result in no major toxicity (Kenny, 1972; Wolinsky and Kenny, 1974; Kenny et al., 1979).
Address reprint requests to Dr. Alexander D. Kenny, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A. Received July 29, 1992; revised and accepted September 15, 1992. Journal of Pharmacological and Toxicological Methods 28, 159-166 (1992) © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
We proposed in 1970 that "carbonic anhydrase is involved in mammalian (rat) bone resorption" (Waite and Kenny, 1970), and subsequent in vitro work has further supported this contention. Acetazolamide, added to bone cultures, inhibits bone resorption induced by PTH, DB-cAMP, calcitriol, PGEz, and forskolin (Hall and Kenny, 1985, 1986, 1987; Gunasekaran et al., 1986; Minkin and Jennings, 1972; Mahgoub and Stem, 1974; Pierce and Waite, 1985; Raisz et al., 1988). Morphological evidence (Gay and Mueller, 1974; Anderson et al., 1982, 1985; Gay et al., 1983; Cao and Gay, 1985; V~ifin~inen and Parvinen, 1983; V~iiin~inen, 1984; Jilka et al., 1985) and other reports (Brown et al., 1990; Pierce et al., 1990; Sly et al., 1983) also support a vital role for CA in bone resorption. Although we suggested in 1970 that "acetazolamide has potential as an effective agent in the treatment of conditions associated with calcium and bone loss" (Waite et al., 1970), this agent, or one of its analogues, has yet to be tested prospectively either in normal subjects or in patients undergoing bone loss with one exception (Gram et al., 1990), The latter group studied the effect of acetazolamide in postmenopausal women for 28 days; no ef1056-8719/92/$5.00
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JPM Vol. 28, No. 3 November 1992:159-166
fects on bone resorption were seen but the drug, as expected, caused a metabolic acidosis that would negate any anticipated inhibition of bone loss. Nevertheless, the CA theory of bone resorption has been implicated as relevant in two other clinical studies (Pierce et al., 1990; Sly et al., 1983). In preparation for a clinical trial of acetazolamide' s effectiveness in reducing bone loss, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum.
Methods
Materials Acetazolamide was a gift of Lederle Laboratories (Wayne, N J). Heat-inactivated horse serum was obtained from Gibco Laboratories [Grand Island, NY; Catalog (Cat.) no. 230-6050]. Human serum was obtained from a volunteer. All other chemicals, including bovine erythrocyte CA II (Sigma C-7500), were obtained from Sigma Chemical Co. (St. Louis, MO). The Centriprep-10 ultrafiltration units with a 15-mL capacity and 10 kD cutoff were obtained from Amicon (Amicon Division, W. R. Grace and Co., Beverly, MA; Cat. no. 4304). The following were obtained from Scientific Products (Scientific Products Division, Baxter Diagnostics, McGaw Park, IL): 3.0-mL polypropylene B-D syringes with luer slip tip (Cat. No. $9519-3S) and 3.5in 17-gauge needles (Cat. no. $9575-76).
Determination of Carbonic Anhydrase Inhibitory Activity Carbonic anhydrase inhibitory activity was determined using an enzymatic assay for CA in the presence of the CA inhibitor such as acetazolamide. Carbonic anhydrase was assayed as described by Hall and Kenny (1985) using a modification of the method of Wilbur and Anderson (1948). The method is based on measuring the rate of COz hydration at 0°C by detecting the pH 6.50 end point with an Orion pH meter (Model 811 ; Orion Research Inc., Boston, MA) using a Fisher electrode (Fisher Scientific, Pittsburgh, PA; Cat. no. 13-620-280). The Wilbur-Anderson (W-A) CA activity unit is defined as [(to/to) - 1] x 10, where tu = time (minutes) of the uncatalyzed reaction and tc = time (minutes) of the catalyzed reaction. Assay buffer. The assay buffer is adjusted to pH 8.2 and consists of 16.7 mM potassium phosphate; 0.005% bromothymol blue; and 0.003% ethylenediaminetetraacetate (EDTA). The buffer may be stored in the refrigerator between assays.
Substrate solution: CO2-saturated water. CO2-saturation is maintained throughout the assay by bubbling CO2 through distilled water on ice; bubbling is started at least 1 hr before use. Acetazolamide standards. Acetazolamide standards are prepared in assay buffer containing 0.9 x 10 -6 M salicylic acid adjusted to pH 8.2 so that the acetazolamide concentrations range from 1.7 × 10-1o M to 1.7 X 10 - 7 M at tenfold intervals. When the 3.0-mL aliquot of acetazolamide standard is diluted to a total of 5.1 mL in the final reaction mixture, these concentrations become 1.0 × 10 -1° M to 1.0 × 10 - 7 M. The salicylic acid concentration is equivalent to that in the diluted serum sample. Carbonic anhydrase solution. Bovine erythrocyte CA II (Sigma C-7500) is prepared in water containing 40 txg/mL of lactic dehydrogenase (LDH) (Sigma L5132) at a concentration to give, when added as a 100IxL aliquot to the assay reaction mixture, a reaction time of approximately 0.50 min in the absence of any inhibitor. A vehicle control containing only 40 txg/mL of LDH is also prepared. The LDH is added to prevent losses of CA II by adherence to the wall of the container.
Final Assay Procedure The final method will be described; modifications used in the course of its development will be indicated where appropriate in the Results section. Serum sample preparation. A 0.5-mL aliquot of human serum is diluted tenfold with 4.5 mL of 10 -6 M salicylic acid dissolved in pH 8.2 assay buffer. The pH of the diluted serum sample is adjusted to 2.5 with 2.0N H 2 5 0 4 . The acidified solution is refrigerated overnight to allow for binding equilibrium with serum proteins and then centrifuged for 15 min at 2000 rpm and 4°C in a refrigerated centrifuge (Damon/IEC CRU 5000). The supernatant is removed and kept on ice for 1-2 hr. The supernatant is placed in an Amicon Centriprep-10 ultrafiltration unit (10 kD cutoff) and centrifuged at 4500 rpm and 4°C for 30 min. A 2.0-mL aliquot of the ultrafiltrate is diluted tenfold with 18.0 mL of the pH 8.2 assay buffer and adjusted to pH 8.2 with 2.0N NaOH; this represents a 100-fold dilution of the original serum sample. Further dilution in assay buffer may be required to fall within the limits of the standard curve. Determination of carbonic anhydrase inhibitory activity. Each determination is assayed in triplicate in 13 x 100 mm disposable glass reaction tubes on ice. The reaction mixture has a total volume of 5.1 mL and consists of three additions: (1) 3.0 mL of either assay buffer, acetazolamide standard, or unknown (serum ultrafiltrate); (2) 100 ~L of either CA solution or its vehi-
I. M. G R E E N E AND A. D. K E N N Y D E T E R M I N A T I O N OF A C E T A Z O L A M I D E IN H U M A N SERUM BY ENZYMATIC ASSAY
Table
161
1. Protocol for Assay of Serum Acetazolamide: Preparation of Tubes Prior to Adding 2.0 mL of Substrate
Tube
Description
Assay Buffer
AZ stda (mL)
Unknownb (mL)
Vehiclec (v~L)
CA Solution d
---
100 --
-100
(wL)
1-3 4-6
Zero inhibition
3.0 3.0
---
7-9
A Z std 10-1o M
--
3.0
--
100
--
A Z std 10 - 9 M
---
3.0 3.0
---
-100
100 --
----
3.0 3.0 3.0
----
-100 --
100 -100
10-12 13-15 16-18 19-21 22-24
A Z std 10 - s M
25-27 28-30
A Z std 10 - 7 M
---
3.0 3.0
---
100 --
-100
31-33 34-36
Unknown serum
---
---
3.0 3.0
100 --
-100
37-39
Unknown serum
---
---
3.0 3.0
100 --
-100
et c e t e r a
Abbreviations: std, s t a n d a r d ; o t h e r a b b r e v i a t i o n s as p e r text. a T h e a c e t a z o l a m i d e c o n c e n t r a t i o n s a r e t h o s e in t h e final r e a c t i o n m i x t u r e o f 5.1 m L ; f o r e x a m p l e , 3.0 m L o f 1.7 × 10 - 9 M b e c o m e s 1.0 × 10 - 9 M w h e n d i l u t e d to a t o t a l v o l u m e o f 5.1 m L . b S e r u m u l t r a t i l t r a t e s a m p l e o r a n y o t h e r s a m p l e to b e a s s a y e d f o r c a r b o n i c a n h y d r a s e i n h i b i t o r y a c t i v i t y .
c T h e v e h i c l e f o r t h e C A s o l u t i o n c o n t a i n s 40 V,g/mL o f L D H in w a t e r . d B o v i n e c a r b o n i c a n h y d r a s e I I s o l u t i o n p r e p a r e d in 40 V,g/mL o f L D H s u c h t h a t it will g i v e a r e a c t i o n t i m e o f a p p r o x i m a t e l y 0.5 m i n in the a b s e n c e of a n y inhibitor.
cle (40 wg/mL LDH); and (3) 2.0 mL of CO2-saturated water substrate. After the first two additions, the mixture is vortexed, the pH electrode is lowered into the reaction tube, and 2.0 mL of substrate solution (CO2saturated water) are injected with a 3.0-mL polypropylene syringe with a 3.5-in 17-gauge needle, using the injection force to thoroughly mix the contents of the reaction tube, at which moment the timer is started. The protocol for preparing the first two additions is presented in Table 1. The time required to reach pH 6.50 is recorded in one-hundredths of a minute. The units are calculated for each pair of three tubes (e.g., tubes 1-3 and 4-6 in Table 1) using the means of each of the triplicate determinations. The units representing uninhibited CA activity (tubes 1-6 in Table 1) and the units calculated for each of the inhibited pairs representing the acetazolamide standard curve (tubes 7-30) are computed using the formula for the Wilbur-Anderson CA activity unit. The uncatalyzed time, tu, and the catalyzed time, to, are represented by the first and second three tubes in each pair respectively (e.g., tubes 19-21 represent tu and tubes 22-24 represent tc in Table 1). The concentrations of the acetazolamide standards are plotted against the percent inhibition of CA activity which is calculated as [ ( U n i t s f o r z e r o inhibition) -
( U n i t s f o r A Z s t a n d a r d ) ] x 100
( U n i t s f o r z e r o inhibition)
present in Figure 1 in which bovine erythrocyte carbonic anhydrase II is used as the standard. The CA inhibitory activity in the unknown serum ultrafiltrate sample, expressed-in terms of acetazolamide concentration, is calculated from the standard curve either graphically or by means of a regression line.
Figure 1. Standard curve for acetazolamide inhibition of carbonic anhydrase II over a concentration range of 10-io to l0 -7 M of acetazolamide using bovine erythrocyte carbonic anhydrase II dissolved in 40 i~g/mLof LDH such that it gave a reaction time of approximately 0.5 min in the absence of any inhibitor. ;>-,
100
r. [... Z
~
80
o ~" p. 60 40
~
20
r.. Q 0
. . . . . . . .
10-10
An example of an acetazolamide standard curve is
i
10-9
. . . .
lllll
10-a
i
i
. . . . . .
10-7 ACETAZOLAMIDE CONCENTRATION (M)
162
JPM Vol. 28, No. 3 1992:159-166
November
¢o
~"
A
~ I
140
UNFILTERED SERUM SERUM FILTRATE 31.5
SERUM FILTPATE 100%
100%
100%
blO0 >
[a.]
-~
. ^ :
8o
20
" Z
I
120
[.-,
3o
uNFILTERED SEPIjM
B
[-,
so
14.9
~
~.
4o 2o
,
-
90
> 0 c.~ F..:l
8O
I
SERUM FILTRATE 74%
70
~
6O
~
50
.< ,-.-1 0
40
-< b"
3O
~
2O 10 0
ND SERUM + 33 ng/ml
SERUM + AZ 660 n g / m l AZ
163
I. M. GREENE AND A. D. KENNY DETERMINATION OF ACETAZOLAMIDE IN HUMAN SERUM BY ENZYMATIC ASSAY
tected .in the ultrafiltrate at either acetazolamide concentration indicating that acetazolamide was highly bound to plasma proteins under these conditions. When the serum sample was analyzed without ultrafiltration, recoveries of 17% and 74% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively. Therefore, ultrafiltration at pH 8.1, in spite of its simplicity, is unacceptable. At this point in developing the method, most subsequent experiments were designed using horse serum because of its ready availability from commercial sources and the absence of the infection hazards associated with human serum.
Table 2. Effect of pH on Recovery of Acetazolamide in the Ultrafdtrate of Horse and Human Serum Samples in the Presence of 10-6 M Salicylic Acid
Effect of Salicylic Acid Concentration on the Carbonic Anhydrase Assay
Effect of Salicylic Acid on Recovery of Acetazolamide from Horse Serum
In the course of the development of the method it was elected to add salicylic acid to the serum sample to determine its effect on protein binding. For this reason it was necessary to determine if and at what concentration salicylic acid interfered with the CA assay. Using a constant concentration of bovine erythrocyte CA II to give a catalyzed time of approximately 0.5 min, salicylic acid was added to the assay buffer to give concentrations ranging from 10 -8 to 10 -3 M at tenfold intervals. The latter are the concentrations in the 3.0 mL of assay buffer; the concentrations in the 5.1-mL final reaction mixture were 0.59 × 10 -8 to 0.59 × 10 - 3 M. The results are presented in Figure 4. Only the higher concentrations (10-* and 10-3 M) exhibited interference: 7% and 22% inhibition, respectively. Therefore, the use of 0.9 × 10 - 7 M salicylic acid in the final method presents no problem with regard to subsequent CA assay.
One milliliter of horse serum was diluted tenfold with 9.0 mL of pH 8.2 assay buffer containing 10 -6 M salicylic acid, and 33 ng of acetazolamide were added; the pH of the diluted sample was 8.1. Seven milliliters of diluted serum, containing 0.9 × 10 -6 M salicylic acid, were ultrafiltered, and a 3.0-mL aliquot of the ultrafiltrate was diluted tenfold with 27.0 mL of pH 8.2 assay buffer. A 3.0-mL aliquot, containing 0.9 × 10 - 7 M salicylic acid and 1 ng of acetazolamide, was assayed as an unknown as described in the Final Assay Procedure. The above procedure was repeated with the addition of 660 ng of acetazolamide instead of 33 ng. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 [Experiment (Exp.) V262]. Recoveries of 18% and 63% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively, indicating that salicylic acid has a definite, but insufficient, effect in decreasing the binding of acetazolamide to serum proteins. Ultrafiltration at pH 8.1 in the presence of 0.9 × 10 - 6 M salicylic acid did decrease protein binding but to a degree which was judged inadequate for a final assay procedure.
Figure 4. Effect of salicylic acid (10 -s to 10 -3 M) on the assay of carbonic anhydrase. ND, not detectable.
Exp. No.
Species
pH
Serum AZ/(ng/mL)
AZ Recovery/(%)
V262
Horse
8.1
V274
Horse
5.0
V278
Horse
2.5
V286
Human
2.5
33 660 33 660 66 660 66 66O
18% 63% 60% 73% 84% 89% 83% 94%
Abbreviations as per text.
25 22%
oF-.
Effect of pH on Recovery of Acetazolamide from Horse Serum
20
o
-== =:l
1,5
t-..t Z t,a rj r.j
10 7%
5
r.J 13., rj
o
i0-o
ND
NO
ND
ND
10-11
10-7
lO-e
10-5
10-4
10-3
lO-Z
SALICYLIC ACID C O N C E N T R A T I O N (M)
Effect of pH 5.0. Three milliliters of horse serum were diluted tenfold with 27.0 mL of pH 8.2 assay buffer containing 10 - 6 M salicylic acid, and 33 ng of acetazolamide were added. The pH of the diluted sample was adjusted to 5.0 with H2SO4., refrigerated overnight, and centrifuged at 2000 rpm at 4°C for 15 min. Seven milliliters of diluted serum at pH 5.0, containing 0.9 x 10 -6 M salicylic acid, were ultrafiltered, and a 3.0-mL aliquot of the ultrafiltrate was diluted tenfold with 27.0 mL of pH 8.2 assay buffer and adjusted to
164
pH 8.2 with NaOH. A 3.0-mL aliquot, containing 0.9 × 10 - 7 M salicylic acid and 1 ng of acetazolamide, was assayed as an unknown as described above in the Final Assay Procedure. The above procedure was repeated with the addition of 660 ng of acetazolamide instead of 33 ng. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V274). Recoveries of 60% and 73% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively, indicating that, in the presence of 0.9 × 10 - 6 M salicyclic acid, pH 5.0 is more effective in decreasing acetazolamide binding to serum proteins than pH 8.1 (Fig. 3). Nevertheless, these recoveries were judged inadequate for a final assay procedure. Effect of pH 2.5. This experiment was similar to the one at pH 5.0 except the pH of the diluted serum sample was adjusted to pH 2.5 (instead of pH 5.0) and 66 and 660 ng of acetazolamide were added; the lower amount was increased from 33 to 66 ng in order to place the final data closer to the center and more reliable part of the standard curve. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V278). Recoveries of 84% and 89% were recorded for the samples containing 66 and 660 ng of acetazolamide, respectively, indicating that, in the presence of 0.9 × 10 - 6 M salicylic acid, pH 2.5 is more effective in decreasing acetazolamide binding to serum proteins than either pH 8.1 or 5.0 [Table 2 (Exp. V262 and V274, respectively)]. These recoveries were judged adequate for a final assay procedure.
JPM Vol. 28, No. 3 November 1992:159-166
sent 330 and 660 ng/mL, respectively, in the original plasma. Of the diluted plasma, 5-mL aliquots were adjusted separately to pH 2.5, and each sample was analyzed as in the Final Assay Procedure. Using the five separate determinations of the acetazolamide concentration, the means and standard deviations were calculated from which the coefficient of variation was derived. The values for the two experiments were respectively as follows: mean, 310 and 594 ng/mL; SDs, ___ 32.4 and _ 55.5; and coefficients of variation, 10.5% and 9.4%.
Analysis of a Serum from a Human Subject on a Low Dose of Acetazolamide A human serum sample, taken from a normal volunteer 1 day after stopping treatment for 1 week with low doses of acetazolamide (150 mg/day administered in three equal doses of 50 mg formulated as a slow-release preparation in a wax matrix by Mission Pharmacal Company, San Antonio, TX) was analyzed by the method described above and found to contain 415 ng/ mL of total acetazolamide. ~ This concentration does not represent steady-state serum concentrations for the 150 rag/day slow-release preparation as the sample was taken 1 day after stopping medication. The therapeutic range for the treatment of glaucoma with recommended doses of the regularly formulated acetazolamide is quoted by Sweeney et al. (1986) to be 5000 to 10,000 ng/mL.
Discussion Recoveries of Acetazolamide from Human Serum at p H 2.5
Precision of the Method
Several methods have been developed for the determination of acetazolamide in human blood samples. These include enzymatic (CA inhibition), high-performance liquid chromatography (HPLC), colorimetric, and electron-capture gas chromatography; their relative merits have been briefly reviewed (Yakatan et al., 1976; Hossie et al., 1980; Chambers et al., 1981). Of these four approaches, only the enzymatic (Maren, 1960; Yakatan et al., 1976) and HPLC (Hossie et al., 1980; Sweeney et al., 1986; Joyce et al., 1989) methods have emerged as the principals. What are the advantages and disadvantages of these two approaches, both of which determine the total (as opposed to the free/ bound) acetazolamide concentrations? Criteria for evaluating methods include simplicity, sensitivity, precision, recovery, and specificity. In general, the HPLC method follows that described
Experiments were conducted to determine the precision of the method. Three milliliters of human serum were diluted with 27 mL of the assay buffer to which 990 or 1980 ng of acetazolamide were added to repre-
The serum sample was kindly supplied by Dr. Charles C. Y. Pak, Department of Internal Medicine, University of Texas Southwestern Medical School at Dallas.
Having shown that recoveries from horse serum were adequate at pH 2.5 and in the presence of 0.9 × 10 -6 M salicylic acid [Table 2 (Exp. V278)], the final experiment was done using the same protocol except substituting human for horse serum. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V286). Recoveries of 83% and 94% were recorded for the samples containing 66 and 660 ng of acetazolamide, respectively, indicating that at pH 2.5 and in the presence of 0.9 x 10 -6 M salicylic acid the recoveries were judged adequate for a final assay procedure.
165
I. M. GREENE AND A. D. KENNY DETERMINATION OF ACETAZOLAMIDE IN HUMAN SERUM BY ENZYMATIC ASSAY
by Hossie et al. (1980). The method involves addition of an internal standard, chlorothiazide, to the diluted plasma sample followed by ethyl acetate extraction, centrifugation to remove the organic phase, repeat of the extraction and centrifugation step, evaporation to dryness under N2, and dissolution of the sample for reverse-phase HPLC. The HPLC method has good precision and recoveries. Coefficients of variation are reported to be between 2.0% and 3.5% (Hossie et al., 1980), 1.1% and 2.5% (Chambers et al., 1981), and 4% and 6% (Joyce et al., 1989). Recoveries of acetazolamide are 94% (Hossie et al., 1980) and 95% (Chambers et al., 1981). Disadvantages are associated with sensitivity and specificity. Sensitivities of 0.5 wg/mL (Hossie et al., 1980; Chambers et al., 1981) and of 0.9 ixg/mL (Joyce et al., 1989) of plasma are claimed. A potential problem with the HPLC approach is the possible interference from therapeutic agents that a subject might be taking concomitantly, not only acetazolamide and the frequently prescribed diuretic, chlorothiazide, but other drugs that may be extracted by ethyl acetate and co-chromatograph with either acetazolamide or chlorothiazide. The enzymatic method described by Maren (1960) and adapted by Yakatan et al. (1976) has the advantages of simplicity and sensitivity. Our method for determining CA activity, described elsewhere (Hall and Kenny, 1985) and in this paper, derives from these two reports. An important modification in our method, in addition to using a pH meter to detect the end point, is the use of ultrafiltration (I0 kD cutoff), which retains contaminating CA but permits filtration of the acetazolamide stripped of protein binding by the addition of salicylic acid and pH adjustment. Sensitivity is excellent. Acetazolamide added to horse serum to give a concentration of 33 ng/mL of serum was readily detectable even at pH 5.0 [Table 2 (Exp. V274)]. This contrasts with the 500 ng/mL (0.5 txg/mL) sensitivity limit of the HPLC method. This sensitivity difference is less important when dealing with normal therapeutic levels of acetazolamide but, in our planned trial, where very low doses of acetazolamide, and, therefore, plasma levels, may be encountered, its importance increases. The rationale for selecting salicylic acid for stripping the acetazolamide bound to protein is the acid's wellknown ability to bind more avidly to serum proteins than other drugs and thereby displace them. Sweeney et al. (1986) reported that, when acetazolamide was administered to two elderly patients receiving high doses of aspirin (1.3 and 3.9 g/day), they exhibited abnormally high plasma total and unbound acetazolamide levels: 20.8 (therapeutic range: 5-10 v.g/mL), and 7.7 I~g/mL, respectively, yielding an unbound percentage of 37% (normal: -3%). In a later study Sweeney et al. (1988) examined the effect of salicylic acid on the
displacement of acetazolamide from binding to human plasma proteins. Acetazolamide was added at a concentration of 8 ixg/mL and the effect of adding salicylic acid at concentrations of 97,201, and 383 ~g/mL was examined. The unbound percentage of acetazolamide increased progressively from 3.3% in the absence of salicylic acid to 5.2%, 11.0%, and 30.0% in the presence in the three increasing concentrations of salicylic acid. Our method measures only the total concentration of serum acetazolamide; it does not determine the unbound fraction. Under normal circumstances the unbound fraction may be assumed to be - 3 % (Sweeney et al., 1988). Only under conditions in which the subject might be receiving significant quantities of a drug or drugs known or suspected to displace acetazolamide from protein binding would this assumption not hold. Taking a medication history of the subject would reveal this potential problem and specific medications could be tested for interference on an ad hoc basis. The method as described would determine all carbonic anhydrase inhibitory activity that would include the acetazolamide being administered as well as any other medication the subject might be receiving which exhibits inhibitory activity. On the surface this may be viewed as a disadvantage due to an apparent lack of absolute specificity for acetazolamide. This relative specificity is actually an advantage in the clinical trials being planned as total carbonic anhydrase inhibitory activity is the desired value if the hypothesis is correct that all carbonic anhydrase inhibitors, not just acetazolamide, retard bone loss.
References Anderson RE, Jee WSS, Woodbury DM (1985) Stimulation of carbonic anhydrase in osteoclasts by parathyroid hormone. Calcif Tissue Int 37:646-650. Anderson RE, Schraer H, Gay CV (1982) Ultrastructural immunocytochemical localization of carbonic anhydrase in normal and calcitonin-treated chick osteoclasts. Anat Rec 204:9-20. Brown GM, Morris CA, Mitnick MA, Insogna KL (1990) Treatment of humoral hypercalcemia of malignancy in rats with inhibitors of carbonic anhydrase. J Bone Miner Res 6:347-354. Cao H, Gay CV (1985) Effects of parathyroid hormone and calcitonin on carbonic anhydrase location in osteoclasts of culture d embryonic chick bone. Experientia 41:1472-1473. Chambers DM, White MH, Kostenbauder HB (1981) Efficient extraction and reversed-phase high-performance liquid chromatography-ultraviolet quantitation of acetazolamide in serum. J Chromatogr 225:231-235. Conaway HH, Waite LC, Kenny AD (1973) Immobilization and bone mass in rats: Effects of parathyroidectomy and acetazolamide. Calcif Tissue Res 11:323-330. Gay CV, Ito MB, Schraer H (1983) Carbonic anhydrase activity in isolated osteoclasts. Metab Bone Dis Rel Res 5:33-39. Gay CV, Mueller WJ (1974) Carbonic anhydrase and osteoclasts: Localization by labeled inhibitor autoradiography. Science 183: 432-434.
166
Gram J, Bollerslev J, Nielsen HK, Larsen HF, Moskilde L (1990) The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women. J Intern Med 228:367-371. Gunasekaran S, Hall GE, Kenny AD (1986) Forskolin-induced bone resorption in neonatal mouse calvaria in vitro. Proc Soc Exp Biol Med 181:438-442. Hall GE, Kenny AD (1985) Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro. Pharmacology 30: 339-347. Hall GE, Kenny AD (1986) Bone resorption induced by parathyroid hormone and dibutyrylcyclic AMP: Role of carbonic anhydrase. J Pharmacol Exp Ther 238:778-782. Hall GE, Kenny AD (1987) Role of carbonic anhydrase in bone resorption: Effect of acetazolamide on basal and parathyroid hormone-induced bone metabolism. Calcif Tissue Int 40:212-218. Hossie RD, Mousseau N, Sved S, Brien R (1980) Quantitation of acetazolamide in plasma by high-performance liquid chromatography. J Pharm Sci 69:348-349. Jilka RL, Rogers JI, Khalifah RG, Vi~niinen HK (1985) Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice. Bone 6:445-449. Joyce PW, Mills KB, Richardson T, Mawer GE (1989) Equivalence of conventional and sustained release oral dosage formulations of acetazolamide in primary open angle glaucoma. Br J Clin Pharmacol 27:597-606. Kenny AD (1985a) Role of carbonic anhydrase in bone: Partial inhibition of disuse atrophy of bone by parenteral acetazolamide. Calcif Tissue Int 37:126-133. Kenny AD (1985b) Role of carbonic anhydrase in bone: Plasma acetazolamide concentration associated with inhibition of bone loss. Pharmacology 31:97-107. Kenny AD (1972) Effect of dietary acetazolamide on plasma electrolytes, bone mass, and renal mineral contents in rats. Proc Soc Exp Biol Med 140:135-139. Kenny AD, Yee JA, Lutherer LO (1979) Inhibition of disuse atrophy of bone in rats by continuous subcutaneous infusion of acetazolamide. Calcif Tissue Int 28:157. Mahgoub A, Stern PH (1974) Carbon dioxide and the effect of parathyroid hormone on bone in vitro. Am J Physiol 226:1272-1275. Maren TH (1960) A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther 130: 26-29. Maren TH (1984) Carbonic anhydrase: The middle years, 1945-1960, and introduction to pharmacology of sulfonamides. In Biology and Chemistry of the Carbonic Anhydrases. Eds., RE Tashian and D Hewett-Emmett. New York: New York Academy of Sciences, pp. 10-17. Minkin C, Jennings JM (1972) Carbonic anhydrase and bone remod-
JPM Vol. 28, No. 3 November 1992:159-166
eling: Sulfonamide inhibition of bone resorption in organ culture. Science 176:1031-1033. Pierce WM Jr, Nardin GF, Fuqua MF, Sabah-Maren E, Stern SH (1990) Effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women. J Bone Miner Res 6: 347-354. Pierce WM Jr, Waite LC (1985) Acetazolamide inhibition of bone resorption: Lack of effect on phosphate release from bone in vitro. Horm Metab Res 13:591-592. Raisz LG, Simmons HA, Thompson W J, Shepard KL, Anderson PS, Rodan GA (1988) Effects of a potent carbonic anhydrase inhibitor on bone resorption in organ culture. Endocrinology 122: 1083-1086. Sly WS, Hewett-Emmett D, Whyte MP, Yu SL, Tashian RE (1983) Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 80:2752-2756. Sweeney KR, Chapron DJ, Brandt JL, Gomolin IH, Feig PU, Kramer PA (1986) Toxic interaction between actazolamide and salicylate: Case reports and a pharmacokinetic explanation. Clin Pharmacol Ther 40:518-524. Sweeney KR, Chapron D J, Kramer PA (1988) Effect of salicylate on serum protein binding and red blood cell uptake of acetazolamide in vitro. J Pharm Sci 77:751-756. Viianiinen HK (1984) Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone, cartilage and giant cell tumor. Histochemistry 81:485-487. V~in~nen HK, Parvinen EK (1983) High active isoenzyme of carbonic anhydrase in rat calvaria osteoclasts. Histochemisty 78: 481-485. Waite LC (1972) Carbonic anhydrase inhibitors, parathyroid hormone and calcium metabolism. Endocrinology 91:1160-1165. Waite LC, Kenny AD (1970) Acetazolamide and calcium metabolism in the rat. In Calcitonin 1969. Eds., S Taylor and GV Foster. London: William Heinemann Medical Books Ltd., pp. 442-450. Waite LC, Volkert WA, Kenny AD (1970) Inhibition of bone resorption by acetazolamide in the rat. Endocrinology 87:1129-1139. Wilbur KM, Anderson NG (1948) Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 176:147-154. Wistrand PJ (1984) The use of carbonic anhydrase inhibitors in ophthalmology and clinical medicine. In Biology and Chemistry of the Carbonic Anhydrases. Eds., RE Tashian and D Hewett-Emmett. New York: New York Academy of Sciences, pp. 609-619. Wolinsky I, Kenny AD (1974) Mechanical strength and mineral content of bone from rats fed acetazolamide. Calcif Tissue Res 16: 257-260. Yakatan GJ, Martin CA, Smith RV (1976) Enzymatic determination of acetazolamide in human plasma. Anal Chim Acta 84:173-177.
Determination of Acetazolamide in Human Serum by Enzymatic Assay Ingrid M. G r e e n e and A l e x a n d e r D. K e n n y Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A.
Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), formerly used as diuretics, still play a role in the treatment of glaucoma, epilepsy, and altitude sickness. There is now hard evidence from both in vitro and in vivo studies in animals that carbonic anhydrase plays a vital function in bone loss. Acetazolamide blocks bone resorption in these experimental models. We have postulated that acetazolamide has potential for the treatment of human conditions associated with bone loss. In preparation for a clinical trial of acetazolamide's effectiveness in this regard, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum. Acetazolamide is stripped from binding to serum proteins by adding 10-6 M salicylic acid and adjusting the pH to 2.5, followed by ultrafiltration through a membrane (10 kD cutoff). The latter permits the free acetazolamide to enter the filtrate but retains any carbonic anhydrase (31 kD) which may contaminate the serum from hemolysis. The carbonic anhydrase inhibitory activity in the filtrate, representing the acetazolamide, is determined in a carbonic anhydrase assay using acetazolamide as the standard. Recoveries of acetazolamide added to human serum ranged from 83% to 94% depending on the concentration. Precision, as judged by the coefficient of variation, was 10.5%.
Keywords: Acetazolamide; Human serum; Method
Introduction Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), were formerly used as diuretics (Maren, 1984) until supplanted by more effective agents. Nevertheless, they still play a role in the treatment of glaucoma, epilepsy, and altitude sickness (Wistrand, 1984). Reports since 1970 have indicated that the CA inhibitor, acetazolamide, has important in vivo and in vitro effects on bone and calcium metabolism. In vivo, acetazolamide inhibits the hypercalcemic responses to parathyroid hormone (PTH) and dibutyryl-cAMP (DBcAMP) (Waite and Kenny, 1970; Waite et al., 1970; Waite, 1972) and partially inhibits disuse osteoporosis in a rat model (Conaway et al., 1973; Kenny, 1985a, b) at doses which result in no major toxicity (Kenny, 1972; Wolinsky and Kenny, 1974; Kenny et al., 1979).
Address reprint requests to Dr. Alexander D. Kenny, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A. Received July 29, 1992; revised and accepted September 15, 1992. Journal of Pharmacological and Toxicological Methods 28, 159-166 (1992) © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
We proposed in 1970 that "carbonic anhydrase is involved in mammalian (rat) bone resorption" (Waite and Kenny, 1970), and subsequent in vitro work has further supported this contention. Acetazolamide, added to bone cultures, inhibits bone resorption induced by PTH, DB-cAMP, calcitriol, PGEz, and forskolin (Hall and Kenny, 1985, 1986, 1987; Gunasekaran et al., 1986; Minkin and Jennings, 1972; Mahgoub and Stem, 1974; Pierce and Waite, 1985; Raisz et al., 1988). Morphological evidence (Gay and Mueller, 1974; Anderson et al., 1982, 1985; Gay et al., 1983; Cao and Gay, 1985; V~ifin~inen and Parvinen, 1983; V~iiin~inen, 1984; Jilka et al., 1985) and other reports (Brown et al., 1990; Pierce et al., 1990; Sly et al., 1983) also support a vital role for CA in bone resorption. Although we suggested in 1970 that "acetazolamide has potential as an effective agent in the treatment of conditions associated with calcium and bone loss" (Waite et al., 1970), this agent, or one of its analogues, has yet to be tested prospectively either in normal subjects or in patients undergoing bone loss with one exception (Gram et al., 1990), The latter group studied the effect of acetazolamide in postmenopausal women for 28 days; no ef1056-8719/92/$5.00
160
JPM Vol. 28, No. 3 November 1992:159-166
fects on bone resorption were seen but the drug, as expected, caused a metabolic acidosis that would negate any anticipated inhibition of bone loss. Nevertheless, the CA theory of bone resorption has been implicated as relevant in two other clinical studies (Pierce et al., 1990; Sly et al., 1983). In preparation for a clinical trial of acetazolamide' s effectiveness in reducing bone loss, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum.
Methods
Materials Acetazolamide was a gift of Lederle Laboratories (Wayne, N J). Heat-inactivated horse serum was obtained from Gibco Laboratories [Grand Island, NY; Catalog (Cat.) no. 230-6050]. Human serum was obtained from a volunteer. All other chemicals, including bovine erythrocyte CA II (Sigma C-7500), were obtained from Sigma Chemical Co. (St. Louis, MO). The Centriprep-10 ultrafiltration units with a 15-mL capacity and 10 kD cutoff were obtained from Amicon (Amicon Division, W. R. Grace and Co., Beverly, MA; Cat. no. 4304). The following were obtained from Scientific Products (Scientific Products Division, Baxter Diagnostics, McGaw Park, IL): 3.0-mL polypropylene B-D syringes with luer slip tip (Cat. No. $9519-3S) and 3.5in 17-gauge needles (Cat. no. $9575-76).
Determination of Carbonic Anhydrase Inhibitory Activity Carbonic anhydrase inhibitory activity was determined using an enzymatic assay for CA in the presence of the CA inhibitor such as acetazolamide. Carbonic anhydrase was assayed as described by Hall and Kenny (1985) using a modification of the method of Wilbur and Anderson (1948). The method is based on measuring the rate of COz hydration at 0°C by detecting the pH 6.50 end point with an Orion pH meter (Model 811 ; Orion Research Inc., Boston, MA) using a Fisher electrode (Fisher Scientific, Pittsburgh, PA; Cat. no. 13-620-280). The Wilbur-Anderson (W-A) CA activity unit is defined as [(to/to) - 1] x 10, where tu = time (minutes) of the uncatalyzed reaction and tc = time (minutes) of the catalyzed reaction. Assay buffer. The assay buffer is adjusted to pH 8.2 and consists of 16.7 mM potassium phosphate; 0.005% bromothymol blue; and 0.003% ethylenediaminetetraacetate (EDTA). The buffer may be stored in the refrigerator between assays.
Substrate solution: CO2-saturated water. CO2-saturation is maintained throughout the assay by bubbling CO2 through distilled water on ice; bubbling is started at least 1 hr before use. Acetazolamide standards. Acetazolamide standards are prepared in assay buffer containing 0.9 x 10 -6 M salicylic acid adjusted to pH 8.2 so that the acetazolamide concentrations range from 1.7 × 10-1o M to 1.7 X 10 - 7 M at tenfold intervals. When the 3.0-mL aliquot of acetazolamide standard is diluted to a total of 5.1 mL in the final reaction mixture, these concentrations become 1.0 × 10 -1° M to 1.0 × 10 - 7 M. The salicylic acid concentration is equivalent to that in the diluted serum sample. Carbonic anhydrase solution. Bovine erythrocyte CA II (Sigma C-7500) is prepared in water containing 40 txg/mL of lactic dehydrogenase (LDH) (Sigma L5132) at a concentration to give, when added as a 100IxL aliquot to the assay reaction mixture, a reaction time of approximately 0.50 min in the absence of any inhibitor. A vehicle control containing only 40 txg/mL of LDH is also prepared. The LDH is added to prevent losses of CA II by adherence to the wall of the container.
Final Assay Procedure The final method will be described; modifications used in the course of its development will be indicated where appropriate in the Results section. Serum sample preparation. A 0.5-mL aliquot of human serum is diluted tenfold with 4.5 mL of 10 -6 M salicylic acid dissolved in pH 8.2 assay buffer. The pH of the diluted serum sample is adjusted to 2.5 with 2.0N H 2 5 0 4 . The acidified solution is refrigerated overnight to allow for binding equilibrium with serum proteins and then centrifuged for 15 min at 2000 rpm and 4°C in a refrigerated centrifuge (Damon/IEC CRU 5000). The supernatant is removed and kept on ice for 1-2 hr. The supernatant is placed in an Amicon Centriprep-10 ultrafiltration unit (10 kD cutoff) and centrifuged at 4500 rpm and 4°C for 30 min. A 2.0-mL aliquot of the ultrafiltrate is diluted tenfold with 18.0 mL of the pH 8.2 assay buffer and adjusted to pH 8.2 with 2.0N NaOH; this represents a 100-fold dilution of the original serum sample. Further dilution in assay buffer may be required to fall within the limits of the standard curve. Determination of carbonic anhydrase inhibitory activity. Each determination is assayed in triplicate in 13 x 100 mm disposable glass reaction tubes on ice. The reaction mixture has a total volume of 5.1 mL and consists of three additions: (1) 3.0 mL of either assay buffer, acetazolamide standard, or unknown (serum ultrafiltrate); (2) 100 ~L of either CA solution or its vehi-
I. M. G R E E N E AND A. D. K E N N Y D E T E R M I N A T I O N OF A C E T A Z O L A M I D E IN H U M A N SERUM BY ENZYMATIC ASSAY
Table
161
1. Protocol for Assay of Serum Acetazolamide: Preparation of Tubes Prior to Adding 2.0 mL of Substrate
Tube
Description
Assay Buffer
AZ stda (mL)
Unknownb (mL)
Vehiclec (v~L)
CA Solution d
---
100 --
-100
(wL)
1-3 4-6
Zero inhibition
3.0 3.0
---
7-9
A Z std 10-1o M
--
3.0
--
100
--
A Z std 10 - 9 M
---
3.0 3.0
---
-100
100 --
----
3.0 3.0 3.0
----
-100 --
100 -100
10-12 13-15 16-18 19-21 22-24
A Z std 10 - s M
25-27 28-30
A Z std 10 - 7 M
---
3.0 3.0
---
100 --
-100
31-33 34-36
Unknown serum
---
---
3.0 3.0
100 --
-100
37-39
Unknown serum
---
---
3.0 3.0
100 --
-100
et c e t e r a
Abbreviations: std, s t a n d a r d ; o t h e r a b b r e v i a t i o n s as p e r text. a T h e a c e t a z o l a m i d e c o n c e n t r a t i o n s a r e t h o s e in t h e final r e a c t i o n m i x t u r e o f 5.1 m L ; f o r e x a m p l e , 3.0 m L o f 1.7 × 10 - 9 M b e c o m e s 1.0 × 10 - 9 M w h e n d i l u t e d to a t o t a l v o l u m e o f 5.1 m L . b S e r u m u l t r a t i l t r a t e s a m p l e o r a n y o t h e r s a m p l e to b e a s s a y e d f o r c a r b o n i c a n h y d r a s e i n h i b i t o r y a c t i v i t y .
c T h e v e h i c l e f o r t h e C A s o l u t i o n c o n t a i n s 40 V,g/mL o f L D H in w a t e r . d B o v i n e c a r b o n i c a n h y d r a s e I I s o l u t i o n p r e p a r e d in 40 V,g/mL o f L D H s u c h t h a t it will g i v e a r e a c t i o n t i m e o f a p p r o x i m a t e l y 0.5 m i n in the a b s e n c e of a n y inhibitor.
cle (40 wg/mL LDH); and (3) 2.0 mL of CO2-saturated water substrate. After the first two additions, the mixture is vortexed, the pH electrode is lowered into the reaction tube, and 2.0 mL of substrate solution (CO2saturated water) are injected with a 3.0-mL polypropylene syringe with a 3.5-in 17-gauge needle, using the injection force to thoroughly mix the contents of the reaction tube, at which moment the timer is started. The protocol for preparing the first two additions is presented in Table 1. The time required to reach pH 6.50 is recorded in one-hundredths of a minute. The units are calculated for each pair of three tubes (e.g., tubes 1-3 and 4-6 in Table 1) using the means of each of the triplicate determinations. The units representing uninhibited CA activity (tubes 1-6 in Table 1) and the units calculated for each of the inhibited pairs representing the acetazolamide standard curve (tubes 7-30) are computed using the formula for the Wilbur-Anderson CA activity unit. The uncatalyzed time, tu, and the catalyzed time, to, are represented by the first and second three tubes in each pair respectively (e.g., tubes 19-21 represent tu and tubes 22-24 represent tc in Table 1). The concentrations of the acetazolamide standards are plotted against the percent inhibition of CA activity which is calculated as [ ( U n i t s f o r z e r o inhibition) -
( U n i t s f o r A Z s t a n d a r d ) ] x 100
( U n i t s f o r z e r o inhibition)
present in Figure 1 in which bovine erythrocyte carbonic anhydrase II is used as the standard. The CA inhibitory activity in the unknown serum ultrafiltrate sample, expressed-in terms of acetazolamide concentration, is calculated from the standard curve either graphically or by means of a regression line.
Figure 1. Standard curve for acetazolamide inhibition of carbonic anhydrase II over a concentration range of 10-io to l0 -7 M of acetazolamide using bovine erythrocyte carbonic anhydrase II dissolved in 40 i~g/mLof LDH such that it gave a reaction time of approximately 0.5 min in the absence of any inhibitor. ;>-,
100
r. [... Z
~
80
o ~" p. 60 40
~
20
r.. Q 0
. . . . . . . .
10-10
An example of an acetazolamide standard curve is
i
10-9
. . . .
lllll
10-a
i
i
. . . . . .
10-7 ACETAZOLAMIDE CONCENTRATION (M)
162
JPM Vol. 28, No. 3 1992:159-166
November
¢o
~"
A
~ I
140
UNFILTERED SERUM SERUM FILTRATE 31.5
SERUM FILTPATE 100%
100%
100%
blO0 >
[a.]
-~
. ^ :
8o
20
" Z
I
120
[.-,
3o
uNFILTERED SEPIjM
B
[-,
so
14.9
~
~.
4o 2o
,
-
90
> 0 c.~ F..:l
8O
I
SERUM FILTRATE 74%
70
~
6O
~
50
.< ,-.-1 0
40
-< b"
3O
~
2O 10 0
ND SERUM + 33 ng/ml
SERUM + AZ 660 n g / m l AZ
163
I. M. GREENE AND A. D. KENNY DETERMINATION OF ACETAZOLAMIDE IN HUMAN SERUM BY ENZYMATIC ASSAY
tected .in the ultrafiltrate at either acetazolamide concentration indicating that acetazolamide was highly bound to plasma proteins under these conditions. When the serum sample was analyzed without ultrafiltration, recoveries of 17% and 74% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively. Therefore, ultrafiltration at pH 8.1, in spite of its simplicity, is unacceptable. At this point in developing the method, most subsequent experiments were designed using horse serum because of its ready availability from commercial sources and the absence of the infection hazards associated with human serum.
Table 2. Effect of pH on Recovery of Acetazolamide in the Ultrafdtrate of Horse and Human Serum Samples in the Presence of 10-6 M Salicylic Acid
Effect of Salicylic Acid Concentration on the Carbonic Anhydrase Assay
Effect of Salicylic Acid on Recovery of Acetazolamide from Horse Serum
In the course of the development of the method it was elected to add salicylic acid to the serum sample to determine its effect on protein binding. For this reason it was necessary to determine if and at what concentration salicylic acid interfered with the CA assay. Using a constant concentration of bovine erythrocyte CA II to give a catalyzed time of approximately 0.5 min, salicylic acid was added to the assay buffer to give concentrations ranging from 10 -8 to 10 -3 M at tenfold intervals. The latter are the concentrations in the 3.0 mL of assay buffer; the concentrations in the 5.1-mL final reaction mixture were 0.59 × 10 -8 to 0.59 × 10 - 3 M. The results are presented in Figure 4. Only the higher concentrations (10-* and 10-3 M) exhibited interference: 7% and 22% inhibition, respectively. Therefore, the use of 0.9 × 10 - 7 M salicylic acid in the final method presents no problem with regard to subsequent CA assay.
One milliliter of horse serum was diluted tenfold with 9.0 mL of pH 8.2 assay buffer containing 10 -6 M salicylic acid, and 33 ng of acetazolamide were added; the pH of the diluted sample was 8.1. Seven milliliters of diluted serum, containing 0.9 × 10 -6 M salicylic acid, were ultrafiltered, and a 3.0-mL aliquot of the ultrafiltrate was diluted tenfold with 27.0 mL of pH 8.2 assay buffer. A 3.0-mL aliquot, containing 0.9 × 10 - 7 M salicylic acid and 1 ng of acetazolamide, was assayed as an unknown as described in the Final Assay Procedure. The above procedure was repeated with the addition of 660 ng of acetazolamide instead of 33 ng. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 [Experiment (Exp.) V262]. Recoveries of 18% and 63% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively, indicating that salicylic acid has a definite, but insufficient, effect in decreasing the binding of acetazolamide to serum proteins. Ultrafiltration at pH 8.1 in the presence of 0.9 × 10 - 6 M salicylic acid did decrease protein binding but to a degree which was judged inadequate for a final assay procedure.
Figure 4. Effect of salicylic acid (10 -s to 10 -3 M) on the assay of carbonic anhydrase. ND, not detectable.
Exp. No.
Species
pH
Serum AZ/(ng/mL)
AZ Recovery/(%)
V262
Horse
8.1
V274
Horse
5.0
V278
Horse
2.5
V286
Human
2.5
33 660 33 660 66 660 66 66O
18% 63% 60% 73% 84% 89% 83% 94%
Abbreviations as per text.
25 22%
oF-.
Effect of pH on Recovery of Acetazolamide from Horse Serum
20
o
-== =:l
1,5
t-..t Z t,a rj r.j
10 7%
5
r.J 13., rj
o
i0-o
ND
NO
ND
ND
10-11
10-7
lO-e
10-5
10-4
10-3
lO-Z
SALICYLIC ACID C O N C E N T R A T I O N (M)
Effect of pH 5.0. Three milliliters of horse serum were diluted tenfold with 27.0 mL of pH 8.2 assay buffer containing 10 - 6 M salicylic acid, and 33 ng of acetazolamide were added. The pH of the diluted sample was adjusted to 5.0 with H2SO4., refrigerated overnight, and centrifuged at 2000 rpm at 4°C for 15 min. Seven milliliters of diluted serum at pH 5.0, containing 0.9 x 10 -6 M salicylic acid, were ultrafiltered, and a 3.0-mL aliquot of the ultrafiltrate was diluted tenfold with 27.0 mL of pH 8.2 assay buffer and adjusted to
164
pH 8.2 with NaOH. A 3.0-mL aliquot, containing 0.9 × 10 - 7 M salicylic acid and 1 ng of acetazolamide, was assayed as an unknown as described above in the Final Assay Procedure. The above procedure was repeated with the addition of 660 ng of acetazolamide instead of 33 ng. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V274). Recoveries of 60% and 73% were recorded for the samples containing 33 and 660 ng of acetazolamide, respectively, indicating that, in the presence of 0.9 × 10 - 6 M salicyclic acid, pH 5.0 is more effective in decreasing acetazolamide binding to serum proteins than pH 8.1 (Fig. 3). Nevertheless, these recoveries were judged inadequate for a final assay procedure. Effect of pH 2.5. This experiment was similar to the one at pH 5.0 except the pH of the diluted serum sample was adjusted to pH 2.5 (instead of pH 5.0) and 66 and 660 ng of acetazolamide were added; the lower amount was increased from 33 to 66 ng in order to place the final data closer to the center and more reliable part of the standard curve. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V278). Recoveries of 84% and 89% were recorded for the samples containing 66 and 660 ng of acetazolamide, respectively, indicating that, in the presence of 0.9 × 10 - 6 M salicylic acid, pH 2.5 is more effective in decreasing acetazolamide binding to serum proteins than either pH 8.1 or 5.0 [Table 2 (Exp. V262 and V274, respectively)]. These recoveries were judged adequate for a final assay procedure.
JPM Vol. 28, No. 3 November 1992:159-166
sent 330 and 660 ng/mL, respectively, in the original plasma. Of the diluted plasma, 5-mL aliquots were adjusted separately to pH 2.5, and each sample was analyzed as in the Final Assay Procedure. Using the five separate determinations of the acetazolamide concentration, the means and standard deviations were calculated from which the coefficient of variation was derived. The values for the two experiments were respectively as follows: mean, 310 and 594 ng/mL; SDs, ___ 32.4 and _ 55.5; and coefficients of variation, 10.5% and 9.4%.
Analysis of a Serum from a Human Subject on a Low Dose of Acetazolamide A human serum sample, taken from a normal volunteer 1 day after stopping treatment for 1 week with low doses of acetazolamide (150 mg/day administered in three equal doses of 50 mg formulated as a slow-release preparation in a wax matrix by Mission Pharmacal Company, San Antonio, TX) was analyzed by the method described above and found to contain 415 ng/ mL of total acetazolamide. ~ This concentration does not represent steady-state serum concentrations for the 150 rag/day slow-release preparation as the sample was taken 1 day after stopping medication. The therapeutic range for the treatment of glaucoma with recommended doses of the regularly formulated acetazolamide is quoted by Sweeney et al. (1986) to be 5000 to 10,000 ng/mL.
Discussion Recoveries of Acetazolamide from Human Serum at p H 2.5
Precision of the Method
Several methods have been developed for the determination of acetazolamide in human blood samples. These include enzymatic (CA inhibition), high-performance liquid chromatography (HPLC), colorimetric, and electron-capture gas chromatography; their relative merits have been briefly reviewed (Yakatan et al., 1976; Hossie et al., 1980; Chambers et al., 1981). Of these four approaches, only the enzymatic (Maren, 1960; Yakatan et al., 1976) and HPLC (Hossie et al., 1980; Sweeney et al., 1986; Joyce et al., 1989) methods have emerged as the principals. What are the advantages and disadvantages of these two approaches, both of which determine the total (as opposed to the free/ bound) acetazolamide concentrations? Criteria for evaluating methods include simplicity, sensitivity, precision, recovery, and specificity. In general, the HPLC method follows that described
Experiments were conducted to determine the precision of the method. Three milliliters of human serum were diluted with 27 mL of the assay buffer to which 990 or 1980 ng of acetazolamide were added to repre-
The serum sample was kindly supplied by Dr. Charles C. Y. Pak, Department of Internal Medicine, University of Texas Southwestern Medical School at Dallas.
Having shown that recoveries from horse serum were adequate at pH 2.5 and in the presence of 0.9 × 10 -6 M salicylic acid [Table 2 (Exp. V278)], the final experiment was done using the same protocol except substituting human for horse serum. The results, expressed in percent recovery of the added acetazolamide, are presented in Table 2 (Exp. V286). Recoveries of 83% and 94% were recorded for the samples containing 66 and 660 ng of acetazolamide, respectively, indicating that at pH 2.5 and in the presence of 0.9 x 10 -6 M salicylic acid the recoveries were judged adequate for a final assay procedure.
165
I. M. GREENE AND A. D. KENNY DETERMINATION OF ACETAZOLAMIDE IN HUMAN SERUM BY ENZYMATIC ASSAY
by Hossie et al. (1980). The method involves addition of an internal standard, chlorothiazide, to the diluted plasma sample followed by ethyl acetate extraction, centrifugation to remove the organic phase, repeat of the extraction and centrifugation step, evaporation to dryness under N2, and dissolution of the sample for reverse-phase HPLC. The HPLC method has good precision and recoveries. Coefficients of variation are reported to be between 2.0% and 3.5% (Hossie et al., 1980), 1.1% and 2.5% (Chambers et al., 1981), and 4% and 6% (Joyce et al., 1989). Recoveries of acetazolamide are 94% (Hossie et al., 1980) and 95% (Chambers et al., 1981). Disadvantages are associated with sensitivity and specificity. Sensitivities of 0.5 wg/mL (Hossie et al., 1980; Chambers et al., 1981) and of 0.9 ixg/mL (Joyce et al., 1989) of plasma are claimed. A potential problem with the HPLC approach is the possible interference from therapeutic agents that a subject might be taking concomitantly, not only acetazolamide and the frequently prescribed diuretic, chlorothiazide, but other drugs that may be extracted by ethyl acetate and co-chromatograph with either acetazolamide or chlorothiazide. The enzymatic method described by Maren (1960) and adapted by Yakatan et al. (1976) has the advantages of simplicity and sensitivity. Our method for determining CA activity, described elsewhere (Hall and Kenny, 1985) and in this paper, derives from these two reports. An important modification in our method, in addition to using a pH meter to detect the end point, is the use of ultrafiltration (I0 kD cutoff), which retains contaminating CA but permits filtration of the acetazolamide stripped of protein binding by the addition of salicylic acid and pH adjustment. Sensitivity is excellent. Acetazolamide added to horse serum to give a concentration of 33 ng/mL of serum was readily detectable even at pH 5.0 [Table 2 (Exp. V274)]. This contrasts with the 500 ng/mL (0.5 txg/mL) sensitivity limit of the HPLC method. This sensitivity difference is less important when dealing with normal therapeutic levels of acetazolamide but, in our planned trial, where very low doses of acetazolamide, and, therefore, plasma levels, may be encountered, its importance increases. The rationale for selecting salicylic acid for stripping the acetazolamide bound to protein is the acid's wellknown ability to bind more avidly to serum proteins than other drugs and thereby displace them. Sweeney et al. (1986) reported that, when acetazolamide was administered to two elderly patients receiving high doses of aspirin (1.3 and 3.9 g/day), they exhibited abnormally high plasma total and unbound acetazolamide levels: 20.8 (therapeutic range: 5-10 v.g/mL), and 7.7 I~g/mL, respectively, yielding an unbound percentage of 37% (normal: -3%). In a later study Sweeney et al. (1988) examined the effect of salicylic acid on the
displacement of acetazolamide from binding to human plasma proteins. Acetazolamide was added at a concentration of 8 ixg/mL and the effect of adding salicylic acid at concentrations of 97,201, and 383 ~g/mL was examined. The unbound percentage of acetazolamide increased progressively from 3.3% in the absence of salicylic acid to 5.2%, 11.0%, and 30.0% in the presence in the three increasing concentrations of salicylic acid. Our method measures only the total concentration of serum acetazolamide; it does not determine the unbound fraction. Under normal circumstances the unbound fraction may be assumed to be - 3 % (Sweeney et al., 1988). Only under conditions in which the subject might be receiving significant quantities of a drug or drugs known or suspected to displace acetazolamide from protein binding would this assumption not hold. Taking a medication history of the subject would reveal this potential problem and specific medications could be tested for interference on an ad hoc basis. The method as described would determine all carbonic anhydrase inhibitory activity that would include the acetazolamide being administered as well as any other medication the subject might be receiving which exhibits inhibitory activity. On the surface this may be viewed as a disadvantage due to an apparent lack of absolute specificity for acetazolamide. This relative specificity is actually an advantage in the clinical trials being planned as total carbonic anhydrase inhibitory activity is the desired value if the hypothesis is correct that all carbonic anhydrase inhibitors, not just acetazolamide, retard bone loss.
References Anderson RE, Jee WSS, Woodbury DM (1985) Stimulation of carbonic anhydrase in osteoclasts by parathyroid hormone. Calcif Tissue Int 37:646-650. Anderson RE, Schraer H, Gay CV (1982) Ultrastructural immunocytochemical localization of carbonic anhydrase in normal and calcitonin-treated chick osteoclasts. Anat Rec 204:9-20. Brown GM, Morris CA, Mitnick MA, Insogna KL (1990) Treatment of humoral hypercalcemia of malignancy in rats with inhibitors of carbonic anhydrase. J Bone Miner Res 6:347-354. Cao H, Gay CV (1985) Effects of parathyroid hormone and calcitonin on carbonic anhydrase location in osteoclasts of culture d embryonic chick bone. Experientia 41:1472-1473. Chambers DM, White MH, Kostenbauder HB (1981) Efficient extraction and reversed-phase high-performance liquid chromatography-ultraviolet quantitation of acetazolamide in serum. J Chromatogr 225:231-235. Conaway HH, Waite LC, Kenny AD (1973) Immobilization and bone mass in rats: Effects of parathyroidectomy and acetazolamide. Calcif Tissue Res 11:323-330. Gay CV, Ito MB, Schraer H (1983) Carbonic anhydrase activity in isolated osteoclasts. Metab Bone Dis Rel Res 5:33-39. Gay CV, Mueller WJ (1974) Carbonic anhydrase and osteoclasts: Localization by labeled inhibitor autoradiography. Science 183: 432-434.
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Gram J, Bollerslev J, Nielsen HK, Larsen HF, Moskilde L (1990) The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women. J Intern Med 228:367-371. Gunasekaran S, Hall GE, Kenny AD (1986) Forskolin-induced bone resorption in neonatal mouse calvaria in vitro. Proc Soc Exp Biol Med 181:438-442. Hall GE, Kenny AD (1985) Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro. Pharmacology 30: 339-347. Hall GE, Kenny AD (1986) Bone resorption induced by parathyroid hormone and dibutyrylcyclic AMP: Role of carbonic anhydrase. J Pharmacol Exp Ther 238:778-782. Hall GE, Kenny AD (1987) Role of carbonic anhydrase in bone resorption: Effect of acetazolamide on basal and parathyroid hormone-induced bone metabolism. Calcif Tissue Int 40:212-218. Hossie RD, Mousseau N, Sved S, Brien R (1980) Quantitation of acetazolamide in plasma by high-performance liquid chromatography. J Pharm Sci 69:348-349. Jilka RL, Rogers JI, Khalifah RG, Vi~niinen HK (1985) Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice. Bone 6:445-449. Joyce PW, Mills KB, Richardson T, Mawer GE (1989) Equivalence of conventional and sustained release oral dosage formulations of acetazolamide in primary open angle glaucoma. Br J Clin Pharmacol 27:597-606. Kenny AD (1985a) Role of carbonic anhydrase in bone: Partial inhibition of disuse atrophy of bone by parenteral acetazolamide. Calcif Tissue Int 37:126-133. Kenny AD (1985b) Role of carbonic anhydrase in bone: Plasma acetazolamide concentration associated with inhibition of bone loss. Pharmacology 31:97-107. Kenny AD (1972) Effect of dietary acetazolamide on plasma electrolytes, bone mass, and renal mineral contents in rats. Proc Soc Exp Biol Med 140:135-139. Kenny AD, Yee JA, Lutherer LO (1979) Inhibition of disuse atrophy of bone in rats by continuous subcutaneous infusion of acetazolamide. Calcif Tissue Int 28:157. Mahgoub A, Stern PH (1974) Carbon dioxide and the effect of parathyroid hormone on bone in vitro. Am J Physiol 226:1272-1275. Maren TH (1960) A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther 130: 26-29. Maren TH (1984) Carbonic anhydrase: The middle years, 1945-1960, and introduction to pharmacology of sulfonamides. In Biology and Chemistry of the Carbonic Anhydrases. Eds., RE Tashian and D Hewett-Emmett. New York: New York Academy of Sciences, pp. 10-17. Minkin C, Jennings JM (1972) Carbonic anhydrase and bone remod-
JPM Vol. 28, No. 3 November 1992:159-166
eling: Sulfonamide inhibition of bone resorption in organ culture. Science 176:1031-1033. Pierce WM Jr, Nardin GF, Fuqua MF, Sabah-Maren E, Stern SH (1990) Effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women. J Bone Miner Res 6: 347-354. Pierce WM Jr, Waite LC (1985) Acetazolamide inhibition of bone resorption: Lack of effect on phosphate release from bone in vitro. Horm Metab Res 13:591-592. Raisz LG, Simmons HA, Thompson W J, Shepard KL, Anderson PS, Rodan GA (1988) Effects of a potent carbonic anhydrase inhibitor on bone resorption in organ culture. Endocrinology 122: 1083-1086. Sly WS, Hewett-Emmett D, Whyte MP, Yu SL, Tashian RE (1983) Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 80:2752-2756. Sweeney KR, Chapron DJ, Brandt JL, Gomolin IH, Feig PU, Kramer PA (1986) Toxic interaction between actazolamide and salicylate: Case reports and a pharmacokinetic explanation. Clin Pharmacol Ther 40:518-524. Sweeney KR, Chapron D J, Kramer PA (1988) Effect of salicylate on serum protein binding and red blood cell uptake of acetazolamide in vitro. J Pharm Sci 77:751-756. Viianiinen HK (1984) Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone, cartilage and giant cell tumor. Histochemistry 81:485-487. V~in~nen HK, Parvinen EK (1983) High active isoenzyme of carbonic anhydrase in rat calvaria osteoclasts. Histochemisty 78: 481-485. Waite LC (1972) Carbonic anhydrase inhibitors, parathyroid hormone and calcium metabolism. Endocrinology 91:1160-1165. Waite LC, Kenny AD (1970) Acetazolamide and calcium metabolism in the rat. In Calcitonin 1969. Eds., S Taylor and GV Foster. London: William Heinemann Medical Books Ltd., pp. 442-450. Waite LC, Volkert WA, Kenny AD (1970) Inhibition of bone resorption by acetazolamide in the rat. Endocrinology 87:1129-1139. Wilbur KM, Anderson NG (1948) Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 176:147-154. Wistrand PJ (1984) The use of carbonic anhydrase inhibitors in ophthalmology and clinical medicine. In Biology and Chemistry of the Carbonic Anhydrases. Eds., RE Tashian and D Hewett-Emmett. New York: New York Academy of Sciences, pp. 609-619. Wolinsky I, Kenny AD (1974) Mechanical strength and mineral content of bone from rats fed acetazolamide. Calcif Tissue Res 16: 257-260. Yakatan GJ, Martin CA, Smith RV (1976) Enzymatic determination of acetazolamide in human plasma. Anal Chim Acta 84:173-177.
