A Rapid, Simple Determination of Sulfur-containing Compounds and Methylmalonic Acid on Plasma using GC-MS-SIM for the Diagnosis of Homocysteinemia

윤혜란,마헤샬타파,Yoon, Hye-Ran,Thapa, Maheshwor The Korean Society of Inherited Metabolic Disease 2015 대한유전성대사질환학회지 Vol.15 No.3

Purpose: If early diagnosis is not made, patients with metabolic disorders as homocystinemia rapidly progress to physical defect or mental retardation resulted in storage of the toxic material into the brain. Therefore, it is necessary to develop an analytical method for a rapid screening and/or correct confirmation diagnosis. Methods: The standard solution of sulfur amino acids spiked plasma was subjected to protein precipitation with methanol, and then consecutively derivatized with trimethylsilyl (TMS) and trifluoroacyl (TFA) and determined by GC-MS. The formation of TMS derivative of the hydroxyl and TFA derivative of amino functional group was performed by BSTFA and MBTFA, respectively. Selective ion monitoring (SIM) mode was used for quantification with selected specific ions. Results: A calibration curve on standard spiked pooled plasma showed a linear relationship with correlation coefficient of 0.9936-0.9992 for all compounds over the range of 0.1-300 ng. The precision and accuracy were within S.D. of 1-15% and RSD of 1-15% for intra-day assay at 2 ng/mL, 15 ng/mL and 30 ng/mL. LOD and LOQ was 0.4 ng/mL and 4 ng/mL respectively. Conclusion: A rapid analytical method was developed to quantify sulfur amino acids and methyl malonic acid, after two-step derivatization procedure with good sensitivity and specificity on human plasma. Advantages of a new method are simplicity and rapidity. The method could be useful for routine analysis, diagnosis of homocysteinemia.

ESI-MS/MS를 이용한 소변 중 Globotriaosylceramide(Gb3)의 정량 및 임상 응용; 패브리병(Fabry) 진단

윤혜란,조경희,강승우,권영주,정춘식,이용수,Yoon, Hye-Ran,Cho, Kyung-Hee,Kang, Seung-Woo,Kwon, Young-Joo,Jeong, Choon-Sik,Lee, Yong-Soo 대한약학회 2007 약학회지 Vol.51 No.2

Measurement of globotriaosylceramide (Gb3, ceramide trihexoside) in urine has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ${\alpha}$-galactosidase A (${\alpha}$-Gal A). The lack of ${\alpha}$-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. A simple, rapid, and highly sensitive analytical method for Gb3 in urine was developed without labor-extensive pre-treatment by electrospray ionization MS/MS (ESI-MS/MS). Only simple 5-fold dilution of urine is necessary for the extraction and isolation of Gb3 in urine. Gb3 in diluted urine was dissolved in dioxane containing C17:0 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. Eight isoforms of Gb3 were completely resolved from urine matrix. C24:0 Gb3 occupied 50% of total Gb3 as a major component in urine. Linear relationship for Gb3 isoforms was found in the range of 0.005${\sim}$5.0 ${\mu}$g/ml. The limit of detection (S/N=5) was 0.005 ${\mu}$g/ml and limit of quantification was 0.05 ${\mu}$g/ml for C24:0 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9598 to 0.9975. This method could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.

유아돌연사증후군과 유전성대사질환

윤혜란,Yoon, Hye-Ran 대한유전성대사질환학회 2013 대한유전성대사질환학회지 Vol.13 No.2

Specific genetic conditions may lead to sudden unexpected deaths in infancy, such as inborn errors of fatty acid oxidation and genetic disorders of cardiac ion channels. The disease may present dramatically with severe hypoketotic hypoglycemia, Reye syndrome or sudden death, typically with a peak of frequency around 3-6 month, whilst neonatal sudden death is quite rare. When undetected, approximately 20-25% of infants will die or suffer permanent neurologic impairment as a consequence of the first acute metabolic decompensation. Meanwhile, the advent of newborn screening for metabolic diseases has revealed populations of patients with disorders of fatty acid oxidation (FAO), the most frequent of which is medium chain acyl-CoA dehydrogenase (MCAD) deficiency. Without this screening, affected individuals would likely succumb to sudden infant death syndrome (SIDS). Here we describe an overview of sudden infant death syndrome and inherited metabolic disorder.

D체와 R체 이성질체 판별과 GC-MS를 이용한 유전성 대사이상질환의 진단법 개발

윤혜란,Yoon, Hye-Ran 대한유전성대사질환학회 2015 대한유전성대사질환학회지 Vol.15 No.2

Since the secretion of specific chiral isomers in urine (or plasma) is very crucial to diagnose some inborn metabolic disorders, clinical application of dual column achiral differential method has been performed for the absolute configuration of chiral compounds. Extracted from the acidified urine with diethyl ether, carboxylic functional group of organic acid (stereoisomers of the volatile) was derivatized with (-)-menthylation or (S)-(+)-3-methyl-2-butylation and followed by O-trifluoroacylation. Each of the enantiomers was accurately separated from the library matched double column (achiral) with a retention index (I). In various inborn metabolic disease urines, absolute chirality was identified correctly in the urine (10 patients) with inborn metabolic disease (including secretion of D, L- lactic acid, D, L-3-hydroxybutyric acid, and D, L-2-hydroxyglutaric acid). In this study, we identified and isolated the volatile diastereomer as a useful diagnostic marker, this successful application to urine specimens may be useful for diagnostic classification of inherited metabolic disorders.

A Sensitive Determination of Plasma Free Fatty Acids Following Tert-butyldimethylsilyl Derivatization using Gas Chromatography-Mass Spectrometry for Screening of Fatty Acid Oxidation Disorders

윤혜란,타파마헤샬,Yoon, Hye-Ran,Thapa, Maheshwor The Korean Society of Inherited Metabolic Disease 2017 대한유전성대사질환학회지 Vol.17 No.2

Purpose: A sensitive gas chromatography mass spectrometry (GC-MS) method was developed for screening of fatty acid oxidation disorders. Methods: The assay utilized a simple protein precipitation with sulfosalicylic acid followed by tert-butyl dimethylsilyl (TBDMS) derivatization of hydroxyl functional group by N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Results: Calibration curves of spiked pooled plasma showed a linear relationship in the range of 0.01 ng -2 mg with correlation coefficient value greater than 0.98. Limits of detection (LOD) and limits of quantification (LOQ) were found in the range of 0.9-8.8 ng and 9-88 ng, respectively. Conclusion: The new developed method might be useful for a rapid, sensitive screening of inherited fatty acid oxidation disorders. In addition, the method expected to be one of the alternative method for screening newborns of metabolic disorders in the laboratories where expensive MS/MS is unavailable.

Evaluation of the Congenital Hypothyroidism for Newborn Screening Program in Korea: A 14-year Retrospective Cohort Study

윤혜란,안선현,이향자,Yoon, Hye-Ran,Ahn, Sunhyun,Lee, Hyangja The Korean Society of Inherited Metabolic Disease 2019 대한유전성대사질환학회지 Vol.19 No.1

목적: 선천성 갑상선기능저하증(Congenital hypothyroidism, CH)은 전세계적으로 출생아 3,000-4,000명 당 1명의 빈도로 발생하는 신생아기에 가장 흔한 내분비질환이다. 본 연구는 1991년부터 2004년까지 남한의 선천성 갑상선기능저하증 발생률의 빈도 및 현재까지 적용되어온 광범위한 신생아 선별검사의 검사방법과 결과를 재평가하기 위해 수행되었다. 방법: 서울 지역에 위치하지만 전국적인 지사를 운영하고 있는 검사기관 서울의과학 연구소(SCL)에서는 서울 외 6개 광역시(부산, 인천, 대구, 대전, 광주, 울산)와 9개도(경기, 강원, 충북, 충남, 전북, 전남, 경북, 경남, 제주)로 한국을 15개 지역으로 분류하였다. 15개 지역으로부터 신생아의 채혈지를 수집하여 서울본원에서 수집하여, TSH 및 유리 T4를 ELISA 검사법으로 신생아선별검사를 수행하였다. SCL 데이터 및 복지부에 보고된 전국적인 양성자수를 검토하였다. 선천성갑상선기능저하증에 대한 신생아선별 검사법의 cut-off 치는 신생아 갑상선자극호르몬(TSH) 측정을 위해서는 20 mIU/L를 유리 T4의 검사를 위해서는 0.8 ng/dL 이하를 사용하였다. TSH 및 유리 T4 ELISA 분석에 기초한 선천성 갑상선기능저하증에 대해 1차 선별검사에서 선천성 갑상선기능저하증 양성은 신생아 671,805명 중 신생아 159명에서 발견되었으며, 이의 발병빈도는 4,225명 중 1명으로 추정되었다 결과: TSH 분석에서 cut-off 20 mIU/ L를 사용했을 때 민감도, 특이도 및 양성 측도(PPV)는 각각 100.0%, 99.7% 및 10.8%였다. 유리 T4 분석을 위해 0.8 ng/dL cut-off를 사용했을 때 민감도, 특이도 및 양성 예측도는 각각 100.0%, 98.5% 및 3.9%였다. 결론: NBS를 통해 얻은 CH의 발병빈도는 2004년 이전에 해외의 여러 국가에서 보고 된 발병빈도와 비교할 만큼 유사하였다. Purpose: Congenital hypothyroidism (CH) is the most common congenital endocrine disorder. The purpose of the present study was to determine the incidence of CH in South Korea during the period from January 1991 to March 2004. Methods: Central data from each city branch of SCL (Seoul Clinical Reference Laboratories) in Yongin, South Korea, was gathered and collectively analyzed. Newborn screening (NBS) for CH was based on measuring the levels of neonatal thyroid stimulating hormone (TSH) and free T4 (a cut-off of 20 mIU/L and less than 0.8 ng/dL, respectively). Results: During the study period, 671,805 live births were screened for CH based on TSH and free T4 ELISA assays. A total of 159 newborns were deemed positive for CH out of 671,805, with a corresponding incidence of 1 in 4,225. When a cut-off of 20 mIU/L was used in TSH assays, the associated sensitivity, specificity, and positive predictive values (PPV) were 100.0%, 99.7%, and 10.8%, respectively. When a cut-off of 0.8 ng/dL in free T4 assays was used, the associated sensitivity, specificity, and PPV were 100.0%, 98.5%, and 3.9%, respectively. Conclusion: CH incidence in South Korea as evidenced by the results of NBS was compared with its incidence and comparable to the other countries prior to 2004.

초고성능액체크로마토그래피-텐덤질량분석법에 의한 혈청 중스핑고신과 스핑고신-1-포스페이트의 동시 분석

나승훈(Seonghoon Na),윤혜란(Hye-Ran Yoon) 대한약학회 2021 약학회지 Vol.65 No.1

Sphingosine (SPH) and sphingosine-1-phosphate (S1P) are emerging as key players in asthma metabolism and in numerous cellular inflammation processes. To identify potential biomarkers of asthma and inflammatory therapeutics, it is essential to determine their levels. Herein, we developed a rapid and sensitive UHPLC-MS/MS method to simultaneously quantify SPH and S1P in human serum using C17-SPH and C17-S1P as internal standards. After methanol precipitation of serum proteins, the supernatants were analyzed by MS/MS performed in the positive ion mode by multiple reaction monitoring. UHPLC analysis (C18 column) was performed using two mobile phase systems (water containing 0.1% formic acid, and 85% acetonitrile containing 0.1% formic acid) within 5 min of the short run. The calibration curves were linear in the range of 0.002-1.5 µg/mL for S1P and SPH with an R 2 greater than 0.9999. The LOD and LOQ were 0.0002 and 0.0004 µg/mL for S1P, and 0.0005 and 0.001 µg/mL for SPH, respectively. The accuracy and precision of the method were in the range of 89.8-100.7% (RSD, 1.5-2.8%) for both SPH and S1P species. We were able to quantify both molecules in serum from healthy and asthmatic patients. These results suggest that SPH and S1P are promising potential biomarkers, and also contribute to the basic data for the construction of an omics-based platform for preventive index prior to asthma diagnosis.

친권에 관한 비교법적 고찰

윤혜란(Yoon, Hye-Ran) 한국가족법학회 2012 가족법연구 Vol.26 No.2

Family laws in our country are unified for the first time and took the systematic form by the enforcement of the Civil Code in 1960, but it was the incomplete law by birth as it was established on the foundation, which applied the Japanese civil laws as is over three times to the base of the clan code system. In addition, the social and economic effects of the rapidly changing modern society might influenced on the family society, first, which might bring in the inevitable changes across all the family laws such as Family Relationship, Marital Relationship, Parents-Child Relationship, Relative Relationship, Inheritance Acts, etc. Through the several times of extensive amendments in family laws, they have been changed from the laws based on the premodern custom to the norms realized the major parts of modern family law philosophy, but its system and contents do not have accommodated all the reality kept changing. Particularly, the violation of the rights, which threaten the mind, body and life of the children and incurs the damage to the property, are frequently committed by the parents, who should protect their children. This might be considered as serious acts of violation against the ‘children's welfare’ and ‘children's best interest’, the basic principals of the family laws, which jeopardize the foundation of family laws. The parental right does not have the nature of dominative rights anymore, which considers the children as the belongings of the parents, but is being changed to the nature of the responsibility and duty of the parents for the children. In these reality, in the event that the exercise of the parental rights would be apparently against the interests of the children, strong restriction on the parental rights against the exercise of such rights becomes necessary to protect the safety and interests of the children. In this respects, this article intented to examine the case of legislation in each country and to find the system to have effectiveness on the exercise and restriction of the parental rights.

혈장 중 케톤체의 옥심-TMS 유도체화 후 GC-MS/SIM을 이용한 분석

윤혜란(Hye-Ran Yoon) 한국분석과학회 2016 분석과학 Vol.29 No.1

탠덤매스에의한 체액 중 Globotriaocylceramide(Gb-3)의 측정을 이용한 한국인 고 위험도군에서의 파브리병 스크리닝

윤혜란(Hye-Ran Yoon) 대한약학회 2011 약학회지 Vol.55 No.1

Fabry disease (FD) is an X-linked inborn error of glycoshpingolipid metabolism resulting from mutation in the enzyme α-galactosidase A gene. The disease is an X-linked lipid storage disorder and the lack of α-Gal A causes an intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb-3). Measurement of Gb-3 in plasma has clinical importance for monitoring after enzyme replacement therapy for confirmed FD patients. Using electrospray ionization MS/MS we had developed, a simple, rapid, and highly sensitive analytical method for Gb-3 in plasma was used for the purpose of screening FD among high risk groups in Korean population. To date, no comprehensive results for FD screening have been performed and reported in Korea. We screened 1,100 outpatients from 13 hospitals (including clinics) to assess the incidence of FD among patients in high risk groups. For patients with borderline level amount of Gb-3, we repeated Gb-3 or performing complementary or confirmative assay with α-Gal A activity and DNA mutaion analysis for confirmation diagnosis. Of 1,100 we diagnosed 3 FD with 2 classical type and 1 carrier (0.27%).