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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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Near-infrared dye IRDye 800CW-labeled butyrylcholinesterase

BChE-IRDye 800CW
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: December 14, 2009.

Chemical name:Near-infrared dye IRDye 800CW-labeled butyrylcholinesterase
Abbreviated name:BChE-IRDye 800CW
Synonym:
Agent Category:Protein
Target:Butyrylcholinesterase
Target Category:Enzyme
Method of detection:Near-infrared fluorescent optical imaging
Source of signal / contrast:IRDye 800CW
Activation:No
Studies:
  • Checkbox Rodents
Click on protein, nucleotide, and gene for more information about butyrylcholinesterase.

Background

[PubMed]

Cholinesterase (ChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine into choline and acetic acid, and thus shuts off neural transmission (1, 2). There are two types of ChE: acetylcholinesterase (AChE, also known as erythrocyte cholinesterase or acetylcholine acetylhydrolase) and butyrylcholinesterase (BChE or BuChE, also known as plasma cholinesterase, pseudocholinesterase, or acylcholine acylhydrolase) (2). Both enzymes are present in cholinergic and noncholinergic tissues as well as in plasma and other body fluids. They differ in substrate specificity, behavior in excess substrate, and susceptibility to inhibitors.

BChE is encoded by the BCHE gene, which is located in humans on chromosome 3q26.1-q26.2 (3). Mutations of the BCHE gene result in various genotypes and phenotypes (4), and some BCHE gene variants, such as atypical, K, J, and H variants, cause reduced activity of BChE. The silent variants lead to total loss of the enzyme activity (0–2% of normal activity). On the other hand, some variants result in increased activity, such as the C5+ variant (combination of BChE with an unidentified protein), the Cynthiana variant (increased amount of BChE than normal level), and the Johannesburg variant (increased BChE activity with normal enzyme level). In the absence of relaxants, there is no known disadvantage for individuals with these variants.

BChE is synthesized in many tissues, including the liver, lungs, heart, and brain. Similar to AChE, a single BCHE gene gives rise to different protein products by alternative splicing in the coding region of the original transcript. This provides a series of diverse but related molecular forms of BChE (G1, G2, and G4). G4 is the predominant isoform in the mature brain. These forms have similar catalytic properties, but they exhibit different cellular and extracellular distributions and non-catalytic activities.

BChE possesses three different enzymatic activities: esterase, aryl acylamidase, and peptidase. The esterase activity of BChE plays an important role in scavenging anti-AChE compounds such as cocaine, heroin, and organophosphate before they reach AChE at physiologically important sites. In the absence of AChE, BChE is believed to serve as a backup to AChE in supporting and regulating cholinergic transmission (5). BChE also inactivates some drugs, e.g., aspirin, amitriptyline, and bambuterol. The aryl acylamidase activity of BChE may be involved in the crosstalk between seratonergic and cholinergic neurotransmission systems, but it is still poorly understood. The peptidase activity of BChE is related to the development and progress of Alzheimer’s disease (AD) which is characterized by a loss of cholinergic neurons (6). In the brains of patients with AD, the level of the membrane-bound G4 form of AChE is selectively reduced by 90% or more in certain regions, while the level of the G1 form is largely unchanged. On the contrary, the G1 form of BChE shows a 30–60% increase, while the G4 form decreases or remains the same as in the normal brain (7). It has been indicated that BChE, which is found in the neuritic plaques and tangles, cleaves the amyloid precursor protein to the β-amyloid protein and helps β-amyloid diffusion to β-amyloid plaques. Abnormal expressions of BChE and AChE have also been observed in human tumors such as meningioma, glioma, acoustic neurinomas, and lung, colon and ovarian cancers (8). However, the relationship is not clear between altered BChE and AChE expressions and tumorigenesis.

Because of the potential diagnostic and therapeutic values, investigators have synthesized various radiolabeled butyrylcholine analogs and tested their feasibilities as tracers for measurement of cerebral BChE activity (9-12). In an attempt to better understand the real-time distribution of BChE from injection site, Duysen and colleagues labeled the BChE directly with fluorescent dye and investigated the BChE pharmacokinetics in BChE knockout mice (13, 14). There is no detectable BChE activity in all tissues and plasma of the BChE−/− mice.

AChE- and BChE-Related Resource Links

Synthesis

[PubMed]

Lyophilized horse BChE containing 1,440 units of BChE activity (1 unit hydrolyzes 1 µmol butyrylthiocholine per min at 25ºC, pH 7.0, when the butyrylthiocholine concentration is 1 mmol) was labeled with IRDye 800CW following the manufacturer’s instruction (13, 14). The fluorescent dye contains an N-hydroxysuccinimide ester reactive group that coupled with the lysines of BChE protein. The conjugate was dialyzed extensively against phosphate-buffered saline to remove excess unreacted dye. The labeled BChE bound 1 dye molecule per mole of protein and lost 6.7% of its initial activity (13). In another report from the same laboratory, the labeled BChE bound 5 dye molecules per mole of protein and lost 60% of its initial activity (14). The labeled BChE was examined on the nondenaturing gel where the primary tetramers with a molecular weight of 340 kDa were confirmed.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

No references are currently available.

Animal Studies

Rodents

[PubMed]

Duysen et al. analyzed the biodistribution of BChE-IRDye 800CW in BChE knockout (BChE−/−) mice after intramuscular injection of either dye alone (n = 2 mice), BChE-IRDye 800CW (n = 7 mice), or native BChE (n = 3 mice) (14). The genetic background of the BChE−/− mice is strain 129Sv. All tissues and plasma of the BChE−/− mice are devoid of BChE activity (5, 15, 16). BChE−/− mice have no distinguishable phenotype. With in vivo optical imaging, the highest fluorescent intensity was observed at the injection site and in the vasculature, bladder, peritoneal cavity, and liver on day 1 after injection of BChE-IRDye 800CW. On day 3, the signal in the liver and bladder was reduced, while the signal in other organs remained comparable to day 1. On day 7, the fluorescent signal in all examined tissues was reduced but still evident. On day 16, the fluorescence was observable only at the site of injection. With ex vivo imaging of frozen sections of the organs prepared on day 3 after injection, strong fluorescence was observed throughout the liver section, at the injection site, in the cortex and medulla of the kidneys, and in the dense connective tissue of the spleen. Muscle, kidneys, fallopian tubes, and lungs had 5–10% of the intensity in the liver, and the spleen, salivary glands, and heart had 1–2% of the intensity in the liver. Measurement of the plasma BChE activity showed that the half-lives of the native BChE and BChE-IRDye 800CW were 67 h and 30 h, respectively. The maximum plasma activity was detected at 30 h after injection for both native BChE (400 units/ml) and BChE-IRDye 800CW (170 units/ml). On day 12, the plasma activity decreased to 1 unit/ml, a level similar to the 1.9 units/ml in wild-type mice. In most organs examined, the maximum enzyme activity was reached on the first day, and a low level of activity was still detected on day 16 after injection. The BChE-IRDye 800CW was metabolized in the liver and excreted through the kidneys in the same manner as the dye. However, the fluorescent intensities did not correlate with the measured BChE activity levels in organs. The brain was free of fluorescence and BChE enzyme activity, indicating that BChE-IRDye 800CW did not cross the blood–brain barrier.

Johnson et al. investigated the distribution of BChE-IRDye 800CW in the brain of BChE−/− mice (n = 16) after intrathecal injection at the L5–L6 interspace (13). The control mice were injected with IRDye 800CW alone. The fluorescent signal in the mouse brain peaked at 6 h for both BChE-IRDye 800CW and dye alone. At 25 h, the signal in the brains of control mice returned to background levels, while the signal in the brains of mice injected with BChE-IRDye 800CW remained at a level equivalent to that observed at 15 min after injection. With ex vivo imaging of organs dissected at 4 h after injection, intense fluorescence was observed in the brain, spinal column, liver, and kidneys of both control mice and mice injected with BChE-IRDye 800CW (n = 3 mice/group). The highest fluorescent signal was observed in the cistern region (prepontine, magna, and supracerebellar) of the brain. At 25 h after injection, mice treated with BChE-IRDye 800CW (n = 5) still had measurable levels of fluorescent signal in the organs in contrast to the organs from control mice (n = 5), which exhibited no detectable signal. The presence of BChE were confirmed with activity assays. There was no detectable BChE activity in the plasma and brain in the control mice (n = 21) at any time point after injection of dye alone and in the treated mice (n = 8) before injection, while the activity was 42 ± 11 units/ml at 4 h (n = 8 mice) and 23 ± 4 units/ml at 25 h (n = 5 mice) after injection of BChE-IRDye 800CW. The BChE activity in the cerebellum, cerebrum, and brain stem of the animals treated with BChE/IRDye 800CW was 0.4–0.7 units/g, without significant difference among them. With confocal laser microscopy of the frozen brain sections prepared at 25 h after injection, the highest fluorescent signal levels were observed in the tissues surrounding the ventricles, cortex, and dentate gyrus, and lower signal levels were observed in the striatum and other hippocampal regions.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Patocka J., Kuca K., Jun D. Acetylcholinesterase and butyrylcholinesterase--important enzymes of human body. Acta Medica (Hradec Kralove) 2004;47(4):215–28. [PubMed: 15841900]
2.
Chiou S.Y., Huang C.F., Hwang M.T., Lin G. Comparison of active sites of butyrylcholinesterase and acetylcholinesterase based on inhibition by geometric isomers of benzene-di-N-substituted carbamates. J Biochem Mol Toxicol. 2009;23(5):303–8. [PubMed: 19827033]
3.
Goodall R. Cholinesterase: phenotyping and genotyping. Ann Clin Biochem. 2004;41(Pt 2):98–110. [PubMed: 15025799]
4.
Primo-Parmo S.L., Bartels C.F., Wiersema B., van der Spek A.F., Innis J.W., La Du B.N. Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am J Hum Genet. 1996;58(1):52–64. [PMC free article: PMC1914969] [PubMed: 8554068]
5.
Li B., Duysen E.G., Saunders T.L., Lockridge O. Production of the butyrylcholinesterase knockout mouse. J Mol Neurosci. 2006;30(1-2):193–5. [PubMed: 17192674]
6.
Geula C., Darvesh S. Butyrylcholinesterase, cholinergic neurotransmission and the pathology of Alzheimer's disease. Drugs Today (Barc) 2004;40(8):711–21. [PubMed: 15510242]
7.
Pepeu G., Giovannini M.G. Cholinesterase inhibitors and beyond. Curr Alzheimer Res. 2009;6(2):86–96. [PubMed: 19355843]
8.
Soreq H., Zakut H. Amplification of butyrylcholinesterase and acetylcholinesterase genes in normal and tumor tissues: putative relationship to organophosphorous poisoning. Pharm Res. 1990;7(1):1–7. [PubMed: 2405380]
9.
Kikuchi T., Zhang M.R., Ikota N., Fukushi K., Okamura T., Suzuki K., Arano Y., Irie T. N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate: a novel radiotracer for quantifying brain butyrylcholinesterase activity by positron emission tomography. Bioorg Med Chem Lett. 2004;14(8):1927–30. [PubMed: 15050629]
10.
Fukuda H., Okamura N. Brain Nerve. 2007;59(3):203–7. [PubMed: 17370645]
11.
Roivainen A., Rinne J., Virta J., Jarvenpaa T., Salomaki S., Yu M., Nagren K. Biodistribution and blood metabolism of 1-11C-methyl-4-piperidinyl n-butyrate in humans: an imaging agent for in vivo assessment of butyrylcholinesterase activity with PET. J Nucl Med. 2004;45(12):2032–9. [PubMed: 15585478]
12.
Virta J.R., Tolvanen T., Nagren K., Bruck A., Roivainen A., Rinne J.O. 1-11C-methyl-4-piperidinyl-N-butyrate radiation dosimetry in humans by dynamic organ-specific evaluation. J Nucl Med. 2008;49(3):347–53. [PubMed: 18287260]
13.
Johnson N.D., Duysen E.G., Lockridge O. Intrathecal delivery of fluorescent labeled butyrylcholinesterase to the brains of butyrylcholinesterase knock-out mice: visualization and quantification of enzyme distribution in the brain. Neurotoxicology. 2009;30(3):386–92. [PMC free article: PMC3864044] [PubMed: 19442823]
14.
Duysen E.G., Lockridge O. Whole body and tissue imaging of the butyrylcholinesterase knockout mouse injected with near infrared dye labeled butyrylcholinesterase. Chem Biol Interact. 2008;175(1-3):119–24. [PubMed: 18486120]
15.
Duysen E.G., Li B., Lockridge O. The butyrylcholinesterase knockout mouse a research tool in the study of drug sensitivity, bio-distribution, obesity and Alzheimer's disease. Expert Opin Drug Metab Toxicol. 2009;5(5):523–8. [PubMed: 19416087]
16.
Li B., Duysen E.G., Lockridge O. The butyrylcholinesterase knockout mouse is obese on a high-fat diet. Chem Biol Interact. 2008;175(1-3):88–91. [PubMed: 18452903]
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