The Laryngoscope Lippincott Williams & Wilkins © 2008 The American Laryngological, Rhinological and Otological Society, Inc. Fluorescent Detection of Rat Parathyroid Glands via 5-Aminolevulinic Acid Scott A. Asher, BS; Glenn E. Peters, MD; Stephen F. Pehler, BS; Kurt Zinn, PhD, DVM; J. Robert Newman, MD; Eben L. Rosenthal, MD Objective: Anatomic identification of parathyroid glands during surgery is challenging and time consum- ing. We sought to determine whether 5-aminolevulinic acid (5-ALA) could produce parathyroid gland fluores- cence to improve their detection in a preclinical model. Methods: Thirty-two rats were administered 0 to 700 mg/kg of 5-ALA by intraperitoneal injection prior to neck exploration under the illumination of a blue light (380 – 440 nm). Tissue fluorescence was assessed at 1, 2, or 4 hours postinjection and then removed for histologic confirmation of parathyroid tissue. Results: Rat parathyroid glands could not be visu- alized under ambient light. At dosages of 300 mg/kg or greater, bilateral parathyroid glands were visualized in 18 of 19 rats using blue light illumination. At dosages less than 300 mg/kg, parathyroid gland fluorescence was de- tected in only 1 of 13 rats. At 2 hours after 5-ALA admin- istration, the net mean intensity of parathyroid gland fluorescence was optimal with a dose of 500 mg/kg. At both 1 and 4 hours after 5-ALA injection, the net mean intensity of parathyroid gland fluorescence was optimal at the highest dose (700 mg/kg) and positively correlated with dosage increases. Conclusion: 5-ALA can be used to selectively detect parathyroid tissue from surrounding tissue in a preclini- cal model. Our data support the use of this technique in the clinical setting. Key Words: 5-aminolevulinic acid, 5-ALA, parathy- roidectomy, protoporphyrin IX, fluorescent detection, parathyroid glands. Laryngoscope, 118:1014 –1018, 2008 INTRODUCTION Identification of parathyroid glands during surgery can be difficult because of their small size, number, ana- tomic variance, proximity to vital structures (e.g., recur- rent laryngeal nerve), and the unreliability of preopera- tive imaging to localize pathology. 1–4 Despite these obstacles, minimally invasive approaches to parathyroid- ectomy and thyroidectomy are becoming the preferred procedures by both patients and surgeons because they are associated with less pain, smaller scars, shorter length of stay, and decreased morbidity. 5 Unfortunately, recent advances have introduced new surgical techniques that continue to be more and more technically challenging to perform. A parathyroid-specific contrast agent that could be used intraoperatively for anatomic visualization would have the potential to shorten operation times and improve outcomes. 5-aminolevulinic acid (5-ALA) is a naturally occur- ring compound present in virtually every cell of the human body. It is formed from glycine and succinyl coenzyme A and represents the first committed inter- mediate compound in hemoglobin biosynthesis. Admin- istration of exogenous 5-ALA overloads the rate- limiting enzyme ferrochelatase responsible for the last step in hemoglobin biosynthesis. The photoreactant molecule, protoporphyrin IX, subsequently accumu- lates. 6 Protoporphyrin IX has been found to have an excitation frequency from 380 to 440 nm, which can be produced by a xenon light source. When protoporphyrin IX relaxes from a triplet state to a singlet state, it releases energy in the form of 635 nm light. Because the concentration of mitochondria (the site of hemoglobin biosynthesis) is higher in malignant tissue, this prop- erty has been exploited because tumor will thus prefer- entially emit more light than surrounding tissue. 7 The parathyroid glands have been hypothesized to also ex- hibit increased fluorescence because they are hyper- metabolic compared with neighboring tissue. 5-ALA has been proven safe and effective for several clinical uses. Topical application is effective for photody- namic therapy of skin malignancies. 8 Fluorescence-guided surgery after oral administration has been used in pa- tients with malignant gliomas, enabling neurosurgeons to From the Department of Surgery, Division of Otolaryngology–Head and Neck Surgery (S.A.A., G.E.P., S.F.P., J.R.N., E.L.R.) and the Department of Medicine (K.Z.), University of Alabama at Birmingham, Birmingham, Ala- bama, U.S.A. Editor’s Note: This Manuscript was accepted for publication January 3, 2008. Presented at the Southern Section Meeting of the Triological Society, January 10, 2008, Naples, Florida, U.S.A. This work was supported by a grant from the American Cancer Society (RSG-06-1006-01-CCE). Send correspondence to Dr. Eben L. Rosenthal, Division of Otolar- yngology, BDB Suite 563, 1808 7th Avenue South, Birmingham, AL 35294- 0012. E-mail: oto@uab.edu DOI: 10.1097/MLG.0b013e3181671b61 Laryngoscope 118: June 2008 Asher et al.: Rat Parathyroid Gland Fluorescence via 5-Aminolevulinic Acid 1014