Acetazolamide and human carbonic anhydrases: retrospect, review and discussion of an intimate relationship

Figures & data

Figure 1. Published and archived articles in PubMed (https://pubmed.ncbi.nlm.nih.gov/) using the search term “carbonic anhydrase acetazolamide” 1952 to date (2023) and (date of search, 26 September 2023). As much as 3504 articles were published with a mean yearly publication rate of 49 articles. The number of articles using the term “carbonic anhydrase” amounts to 19,193. Inset shows the chemical structure of non-protonated AZM (N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide).

Figure 2. Time course of (A) the creatinine concentration in the urines samples collected in the study, of (B) the creatinine-corrected excretion rate of AZM (AZM), of (C) the creatinine-corrected excretion rates of nitrate and nitrite, and of (D) the urinary nitrate-to-urinary nitrite (U_NOxR) in the respective urine samples without (blue) and with acidification (red) using 20 wt% acetic anhydride. Acidification of urine is required for the accurate measurement of nitrite¹², but not for other analytes including nitrate, creatinine, AZM and amino acids. AUC, area under the curve. AZM, acetazolamide.

Table 1. Urinary creatinine-corrected excretion rates (µmol analyte/mmol creatinine) of nitrite, nitrate and malondialdehyde (acidified urine samples), amino acids and some of their metabolites (non-acidified urine samples) before (0 h) and until 5 h of AZM intake (a 250-mg tablet Acemit^®; Medphano, Germany) by a healthy female volunteer.

Time (h)	0	0.5	1	1.5	2	3	4	5
Nitrite	1.9	2.05	2.93	1.64	1.43	1.61	1.14	0.57
Nitrate	400	490	620	460	400	400	330	240
MDA	1.72	1.00	0.52	0.50	0.41	0.37	0.31	0.30
ADMA	2.7	3.84	3.3	3.11	3.68	3.19	2.73	3.77
SDMA	3.07	4.61	4.18	3.64	3.68	3.51	3.19	4.04
DMA	22.9	32.1	29.9	28.2	27.3	26.6	24.6	34.7
Ala	42.9	56.6	39.2	24.5	22.0	21.6	17.8	18.8
Thr	20.6	31.2	23.7	18.6	17.1	17.6	14.4	17.0
Gly	111	174	134	135	118	123	135	201
Val	6.48	11.5	9.82	9.41	9.16	7.55	6.85	8.33
Ser	40.8	64.4	67.0	37.5	37.1	41.1	32.2	34.4
Sarcosine	0.92	1.37	0.98	1.00	0.97	1.10	0.87	1.18
Leu + Ile	9.27	22.6	28.2	29.3	22.0	26.3	31.7	54.1
GAA	14.3	19.9	18.2	14.1	11.0	11.4	7.78	6.56
Asn + Asp	16.2	25.1	19.9	18.8	17.4	17.7	15.3	18.9
Hydroxy-Pro	0.51	0.85	0.85	1.22	0.55	0.77	0.63	0.63
Pro	2.09	3.33	2.89	1.81	2.62	2.42	2.06	2.45
Gln + Glu	99.5	130	106	91.8	85.5	77.4	71.7	112
Met	12.6	19.4	19.1	12.5	11.7	11.5	9.37	10.5
Orn + Cit	3.29	3.53	2.76	2.52	3.03	2.56	2.02	2.87
Phe	5.55	9.31	9.25	8.47	8.26	7.53	6.82	8.84
Tyr	11.6	17.3	15.5	14.7	14.4	13.8	12.2	15.4
Lys	582	809	668	397	335	293	193	191
Arg	2.15	3.29	2.69	2.35	3.00	2.08	1.68	2.21
hArg	0.15	0.22	0.22	0.20	0.17	0.13	0.10	0.12
Trp	8.78	12.1	10.2	9.09	9.41	9.91	8.04	9.83

Table 2. Results of the time series analysis (ARIMA) with respect to the AZM-induced changes in creatinine-corrected urinary excretion rate of the analytes alongside their chemical structures and electrical charge at physiological pH values of plasma and urine.

Analyte	r^2	p	Structure	Net charge
Nitrite	0.335	0.13		Negative
Nitrate	0.471	0.06		Negative
Malondialdehyde (MDA)	0.687	0.01		Negative
Asparagine (Asn)	0.137	0.37		Neutral
Aspartate (Asp)				Negative
Glutamine (Gln)	0.202	0.26		Neutral
Glutamate (Glu)				Negative
Alanine (Ala)	0.745	0.006		Neutral
Threonine (Thr)	0.505	0.048		Neutral
Glycine (Gly)	0.1	0.37		Neutral
Valine (Val)	0.09	0.46		Neutral
Serine (Ser)	0.37	0.107		Neutral
Sarcosine (Sarc)	0.004	0.88		Neutral
Leucine (Leu)	0.64	0.017		Neutral
Isoleucine (Ile)				Neutral
Proline (Pro)	0.069	0.5		Neutral
Methionine (Met)	0.461	0.06		Neutral
Phenylalanine (Phe)	0.014	0.78		Neutral
Tyrosine (Tyr)	0.05	0.87		Neutral
Tryptophan (Trp)	0.107	0.424		Neutral/positive
Ornithine (Orn)	0.424	0.08		Positive
Citrulline (Cit)				Neutral
Guanidinoacetate (GAA)	0.745	0.006		Positive
Lysine (Lys)	0.799	0.003		Positive
Arginine (Arg)	0.237	0.22		Positive
Homoarginine (hArg)	0.489	0.053		Positive
Symmetric dimethylarginine (SDMA)	0.017	0.7		Positive
Asymmetric dimethylarginine (ADMA)	0.02	0.7		Positive
Dimethylamine (DMA)	0.067	0.5		Positive
Creatinine	n.a.	n.a.		Positive
Acetazolamide (AZM)	n.a.	n.a.		Positive

n.a.: not applicable.

Table 3. Literature pharmacokinetic data of acetazolamide in humans.

Reference	Plasma (PL), serum (SE), erythrocytes (ER), blood (BL)				Urine			Administration and dose	Method	Remarks
1st Author et al.	Tmax	Cmax	t½β	t½abs, tlag	Tmax	Nmax, Cmax	t½β
Ritschel et al.^9^,^10	1–2 h	45 µM	4–6 h PL 9–12 h BL	0.7–1.0 h	n.r.	200 mg	9–12 h	a single 250 mg AZM tablet	HPLC
Chapron et al.^16	1.2 h (ER)	227 µM PL 173 µM ER	2.5 h PL	n.a.	n.a.	0.40 mg/min (at 1h) 0.05 mg/min (at 7h)	2 h	5 mg/kg i.v.	HPLC	Altitude non-linear
Yano et al.^17	2 h	55 µM PL	7 h PL	1.1 h, 0.5 h	n.r.	n.r.	n.r.	125–500 mg b.i.d. oral	HPLC	IOP
Tsikas et al. (this work)	n.m.	n.m.	n.m.	n.m.	2 − 5 h	64 µM/mM/min		a single 250 mg AZM tablet	GC-MS	Healthy
Busardò et al.^31	n.r.	n.r.	n.r.	n.r.	1 h	260 µM/mM	3 h	a single 250 mg AZM tablet	LC-MS/MS	Glaucoma
Hossie et al.^33	1.0 h 2.5 h	6.3 µM 5.9 µM	6 h 6 h	n.r.	n.m.	n.m.	n.m.	1) single 250 mg AZM is solution 2) a 500-mg sustained-release tablet	HPLC	Healthy
Campíns-Falcó et al.^34	n.m.	n.m.	n.m.	n.m	n.r.	35 µM, 19 µM, 2.5 µM at 8 h, 24 h, 48	9 h	a single 250 mg AZM tablet	HPLC	Healthy
Chapron et al.^32	n.r.	227 µM 0 h 24 µM 5 h	n.r.	n.a.	n.r.	320 mg, 0.9 µmol/min	n.r	320 mg AZM i.v.	HPLC	Healthy
Hampson et al.^35	1 h	4.5 µM	24 h	n.r.	n.r.	512–1024 µg/h	16 h	15 mg AZM oral	LC-MS/MS	Healthy
Yacatan et al.^37	0.2 h 8 h	23 µM 82 µM	6 h 10 h	n.r.	n.m.	n.m.	n.m.	250 mg AZM different tablets	Enzymatic	Healthy
Straughn et al.^38	0.5 h 3.0 h	4.5 µM 9.5 µM	3.8 h 9.8 h	n.r.	n.m.	n.m.	n.m	275 mg AZM different tablets	Enzymatic	Healthy

n.r., not reported; n.m., not measured

Table 4. Literature pharmacodynamics data of acetazolamide in humans.

Reference	Urine			Plasma, serum, erythrocytes			Administration and dose	Remark
1st Author et al.	HCO3^−	Na^+	pH	HCO3^−	Na^+	pH
Ritschel et al.^9^,^10	n.r.	n.r.	4.5–7.8 4.2–6.9 3.1–6.7	n.r.	n.r.	n.r.	a single 250 mg AZM tablet
Tsikas, Chobanyan^11	40 mM/mM 25 mM/mM	n.r.	5.5–7.5 5.1–7.6	n.r.	n.r.	n.r.	6.3 mg/kg 5.0 mg/kg (retard capsule)	GC-MS
Chobanyan et al.^12	n.r.	n.r	5.1–7.5 (1h)	n.r.	n.r.	n.r.	5.0 mg/kg	GC-MS
Leaf et al.^24			5.5–7.5			7.38–7.28	250 mg 4x daily	Tolerance
Hanley abd Platts^25	3–140 mM	n.r.		25–19 mM	n.r.	7.40–7.35	e.g. 250 mg 4xdaily 3–4 days	Self-limiting effect
Tsikas et al. (this work)	n.m.	n.m.	7.1–7.65	n.m.	n.m.	n.m.	a single 250 mg AZM tablet	Healthy
Galdston 1955^53	16–244 mM 0.1–15 mM	105–450 mM 13–197 mM	7.26–7.65 5.56–6.79	19–35 mM 21–44 mM	n.m.	7.21–7.39 7.26–7.43	single AZM dose (2.2–6.5 mg/kg); long term; drug-free	Patients
Seldin et al.^66	68–121–118 mmHg		5.94–6.82–7.0 (max.)	11.4–24.8 mM		7.23–7.56	single i.v. injection of 250 mg AZM	Healthy young men
Martens et al.^71	AZM improves decongestive response over the entire range of HCO3^− levels; the treatment response is magnified in patients with baseline elevated HCO3^− levels by specifically counteracting diuretic resistance							519 patients with acute heart failure
Berthelsen et al.^74	1) 1–357 µmol/min 2) 2–578 µmol/min	n.r.	1) 5.35–7.60 2) 6.33–7.66	n.r.	n.r.	2) 7.47–7.43	1) increasing cumulative i.v. doses of AZM (0–10 mg/kg) 2) single i.v. AZM dose (5 mg/kg)	Critically ill
Lichter et al.^84	Tolerance rates (1) vs 2): 95 vs 100% after 1 week 63 vs 84% after 6 weeks 26 vs 58% beyond 6 weeks						1) 250 mg AZM tablets 4xdaily, 1 and 6 weeks 2) 500 mg AZM Sequels 2xdaily, 1 and 6 weeks	Glaucoma Tolerance

n.r.: not reported; n.m.: not measured

Figure 3. Simplified schematic of the role of carbonic anhydrase isoforms CA II (cytosolic) and CA IV (membrane-bound) and some transporters in the proximal tubule of the healthy kidney on the reabsorption of Na⁺, HCO₃^–, water, nitrite, nitrate, malondialdehyde (MDA) and zwitterionic amino acids (⁺AA^-) under two different conditions: (A) Under physiological conditions, i.e. in the absence of AZM; (B) in the presence of AZM; AZM was taken orally at the time point zero as indicated by the vertical red coloured arrow; the time-course of the concentration of AZM in the urine are shown in addition to that of bicarbonate and sodium ions as well as of the urinary pH. AA, amino acid; AQP, aquaporin; GM, glomerular membrane; NHE₃, sodium hydrogen exchanger; kNBC1, sodium bicarbonate transporter. It is assumed that CO₂ is transferred by aquaporin and protein gas channels ⁹⁸. The thickness of arrows indicate the status of the activity of carbonic anhydrase and the transporters. In case of ingestion of a sustained (retard) AZM, the maximal concentrations may be different and longer lasting ^11–13. For more details, see the text.

Chobanyan-Jürgens K, Schwarz A, Böhmer A, Beckmann B, Gutzki F-M, Michaelsen JT, Stichtenoth DO, Tsikas D. Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite. Nitric Oxide. 2012;26(2):126–131.

 PubMed Web of Science ®Google Scholar

Ritschel WA, Paulos C, Arancibia A, Agrawal MA, Wetzelsberger KM, Luecker PW. Urinary excretion of acetazolamide in healthy volunteers after short- and long-term exposure to high altitude. Methods Find Exp Clin Pharmacol. 1998;20(2):133–137.

 PubMedGoogle Scholar

Ritschel WA, Paulos C, Arancibia A, Agrawal MA, Wetzelsberger KM, Lücker PW. Pharmacokinetics of acetazolamide in healthy volunteers after short- and long-term exposure to high altitude. J Clin Pharmacol. 1998;38(6):533–539.

 PubMed Web of Science ®Google Scholar

Chapron DJ, Sweeney KR, Feig PU, Kramer PA. Influence of advanced age on the disposition of acetazolamide. Br J Clin Pharmacol. 1985;19(3):363–371.

 PubMed Web of Science ®Google Scholar

Yano I, Takayama A, Takano M, Inatani M, Tanihara H, Ogura Y, Honda Y, Inui K. Pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure elevation. Eur J Clin Pharmacol. 1998;54(1):63–68.

 PubMed Web of Science ®Google Scholar

Busardò FP, Lo Faro AF, Sirignano A, Giorgetti R, Carlier J. In silico, in vitro, and in vivo human metabolism of acetazolamide, a carbonic anhydrase inhibitor and common “diuretic and masking agent” in doping. Arch Toxicol. 2022;96(7):1989–2001.

 PubMed Web of Science ®Google Scholar

Hossie RD, Mousseau N, Sved S, Brien R. Quantitation of acetazolamide in plasma by high-performance liquid chromatography. J Pharm Sci. 1980;69(3):348–349.

 PubMed Web of Science ®Google Scholar

Campíns-Falcó P, Herráez-Hernández R, Sevillano-Cabeza A. Application of column-switching techniques to the determination of medium polarity drugs: determination of acetazolamide in urine. J Chromatogr B Biomed Appl. 1994;654(1):85–90.

 PubMedGoogle Scholar

Chapron DJ, White LB. Determination of acetazolamide in biological fluids by reverse-phase high-performance liquid chromatography. J Pharm Sci. 1984;73(7):985–989.

 PubMed Web of Science ®Google Scholar

Hampson AJ, Babalonis S, Lofwall MR, Nuzzo PA, Krieter P, Walsh SL. A pharmacokinetic study examining acetazolamide as a novel adherence marker for clinical trials. J Clin Psychopharmacol. 2016;36(4):324–332.

 PubMed Web of Science ®Google Scholar

Yakatan GJ, Frome EL, Leonard RG, Shah AC, Doluisio JT. Bioavailability of acetazolamide tablets. J Pharm Sci. 1978;67(2):252–256.

 PubMed Web of Science ®Google Scholar

Straughn AB, Gollamudi R, Meyer MC. Relative bioavailability of acetazolamide tablets. Biopharm Drug Dispos. 1982;3(1):75–82.

 PubMed Web of Science ®Google Scholar

Tsikas D, Chobanyan-Jürgens K. Quantification of carbonate by gas chromatography-mass spectrometry. Anal Chem. 2010;82(19):7897–7905.

 PubMed Web of Science ®Google Scholar

Leaf A, Schwartz WB, Relman AS. Oral administration of a potent carbonic anhydrase inhibitor (diamox). I. Changes in electrolyte and acid-base balance. N Engl J Med. 1954;250(18):759–764.

 PubMed Web of Science ®Google Scholar

Hanley T, Platts MM. Observations on the metabolic effects of the carbonic anhydrase inhibitor diamox: mode and rate of recovery from the drug’s action. J Clin Invest. 1956;35(1):20–30.

 PubMed Web of Science ®Google Scholar

Galdston M. Respiratory and renal effects of a carbonic anhydrase inhibitor (diamox) on acid-base balance in normal man and in patients with respiratory acidosis. Am J Med. 1955;19(4):516–532.

 PubMed Web of Science ®Google Scholar

Seldin DW, Portwood RM, Rector FC, Jr, Cade R. Characteristics of renal bicarbonate reabsorption in man. J Clin Invest. 1959;38(10 Pt 1–2):1663–1671.

 PubMed Web of Science ®Google Scholar

Martens P, Verbrugge FH, Dauw J, Nijst P, Meekers E, Augusto SN, Ter Maaten JM, Heylen L, Damman K, Mebazaa A, et al. Pre-treatment bicarbonate levels and decongestion by acetazolamide: the ADVOR trial. Eur Heart J. 2023;44(22):1995–2005.

 PubMed Web of Science ®Google Scholar

Berthelsen P, Gøthgen I, Husum B, Jacobsen E. Dissociation of renal and respiratory effects of acetazolamide in the critically ill. Br J Anaesth. 1986;58(5):512–516.

 PubMed Web of Science ®Google Scholar

Lichter PR, Newman LP, Wheeler NC, Beall OV. Patient tolerance to carbonic anhydrase inhibitors. Am J Ophthalmol. 1978;85(4):495–502.

 PubMed Web of Science ®Google Scholar

Itel F, Al-Samir S, Öberg F, Chami M, Kumar M, Supuran CT, Deen PM, Meier W, Hedfalk K, Gros G, et al. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels. Faseb J. 2012;26(12):5182–5191.

 PubMed Web of Science ®Google Scholar