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Development of an ELISA for detection of mitragynine and its metabolites in human urine

Lee MJ, Ramanathan S, Mansor SM, Tan SC

Anal Biochem, 2020 06 15;599:113733.
PMID: 32302607 DOI: 10.1016/j.ab.2020.113733

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.
Mastoid Cyst in a Child

Lee MJ, Lim CC, Ling SW, Devesayaham PR

Indian J Otolaryngol Head Neck Surg, 2023 Dec;75(4):4020-4023.
PMID: 37974688 DOI: 10.1007/s12070-023-04047-y

Mastoid cysts are a rare condition that occurs either congenitally or secondary to chronic otological diseases with poor ventilation of the middle ear and mastoid cavity. In this presenting case, the authors report a case of secondary mastoid cyst with evidence of bony destruction and fistulous tract formation into adjacent structures. Concurrently, we reviewed other similar cases reported in the literature. A 12-year-old Malay boy with partially controlled allergic rhinitis presented with right-sided postauricular swelling and intermittent fever for three months. Examination revealed a non-tender bony swelling over the mastoid area. High-resolution computed tomography of the temporal bone revealed a large bony lytic lesion at the inferior aspect of the mastoid bone. Multidirectional fistulous tracts towards the splenius capitis muscle were seen extending from the lesion. Right mastoid exploration was done and revealed an infected mastoid cyst. Complete removal of the cyst wall and saucerization of the mastoid cavity were performed. Our patient showcases the potential of mastoid cysts to cause bony erosion, fistulation and abscess formation. Complete removal of mastoid cysts is therefore advocated to prevent its potential complications and unwarranted recurrence.
Effect of maternal age, birth weight and infant sex on total nucleated cell (TNC) count and volume of umbilical cord blood (UCB) collected

Choong SN, Ng YK, Kamalan A, Saraswathy S, Goh EH, Lee MJ, et al.

Med J Malaysia, 2008 Jul;63 Suppl A:55-6.
PMID: 19024981

This study evaluates the effect of maternal age, birth weight and infant sex on two main UCB parameters for use and long-term cryopreservation: TNC and volume. Data from 1000 UCB units were collected and analyzed in this study. The results indicate that TNC is correlated to infant birth weight and sex but not maternal age at delivery. Volume is only correlated to birth weight but not maternal age and infant sex.
Method validation in quantitative analysis of phase I and phase II metabolites of mitragynine in human urine using liquid chromatography-tandem mass spectrometry

Lee MJ, Ramanathan S, Mansor SM, Yeong KY, Tan SC

Anal Biochem, 2018 02 15;543:146-161.
PMID: 29248503 DOI: 10.1016/j.ab.2017.12.021

A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.
Acquired Hemophilia A: Rare Cause of Upper Airway Hematoma and a Literature Review

Lim CC, Ling SW, Vijayan KV, Lee MJ, Devesayaham PR

Indian J Otolaryngol Head Neck Surg, 2023 Dec;75(4):3886-3888.
PMID: 37974717 DOI: 10.1007/s12070-023-03943-7

BACKGROUND: Acquired hemophilia A (AHA) is a rare blood disorder with high morbidity and even mortality as severe bleeding can occur in up to 90% of affected patients. Unlike congenital hemophilia which presents with intra-articular bleeding, acquired hemophilia causes bleeding into the skin, muscle, mucous membranes and soft tissues.
CASE PRESENTATION: We report an unusual case of upper airway hematoma in a 61-year-old man who presented with acute onset dysphagia and shortness of breath. There were bruises on his neck and blood clots on the floor of mouth when he was examined. Endoscopic examination revealed an extensive laryngeal hematoma. A prolonged activated partial thromboplastin time (APTT) prompted us to investigate for factor VIII deficiency leading to the diagnosis of AHA. He recovered completely after a treatment regime instituted by the hematology team without suffering any grave debilitating events.
CONCLUSION: AHA with laryngeal hematoma is a rare condition with only a handful of cases reported. Although a life-threatening disease, it is easily reversed with early recognition and administration of medical therapy involving the hematology team.
Screening for G71R mutation of the UGT1A1 gene in the Javanese-Indonesian and Malay-Malaysian populations

Sutomo R, Talib NA, Yusoff NM, Van Rostenberghe H, Sadewa AH, Sunarti, et al.

Pediatr Int, 2004 Oct;46(5):565-9.
PMID: 15491385

There are significant differences in the prevalence and severity of neonatal jaundice among various populations. Recently, it has been reported that a mutation of the UGT1A1 gene, glycine to arginine at codon 71 (G71R), is related to the development of neonatal jaundice in East Asian populations. However, whether the G71R mutation contributes to the high incidence of neonatal jaundice in different Asian populations remains unknown. The authors screened for this mutation in the Javanese-Indonesian and Malay-Malaysian populations.
Fulltext Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1

Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, et al.

Autophagy, 2021 Jan;17(1):1-382.
PMID: 33634751 DOI: 10.1080/15548627.2020.1797280

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.