조혈모세포이식 환자에서 거대세포바이러스의 정량적 검출을 위한 뇨침사의 적절성

강민구 전남대학교 대학원 2015 국내석사

Background: Urine is an important source for the detection of infections caused by cytomegalovirus (CMV) in stem cell transplant patients. Currently, there is no agreement about the type of urine specimen. In order to investigate which is the better specimen type for quantitative detection of CMV, the results from urine supernatant and sediment in same patients were compared. Materials and methods: Seventy urine specimens were collected from patients with hematological disorders or solid tumors. After performing shell vial culture, residual urine specimens were centrifuged. Then, 10 mL of each urine supernatant and sediment were taken and immediately frozen at -70°C. Afterwards, archived urine specimens were thawed at room temperature and CMV-quantitative PCR was performed on both the supernatant and sediment fraction of urine. The results from each patient were reviewed for CMV antigenemia, blood shell vial culture, CMV-IgM or IgG, and clinical symptoms. Results: CMV-qPCR result for the urine sediment fraction revealed a significant difference (p = 0.012) between the active CMV infection group and the latent CMV infection group. In addition, receiver operating characteristic curve for active CMV infection revealed that CMV-qPCR by using urine sediment produced more accurate results rather than urine supernatant. Conclusions: These findings suggest that the sediment fraction of urine is a more suitable specimen in CMV-qPCR testing.

Banana Peel Ash as An Additive to Derive Struvite Fertilizer from Source-separated Urine

박은하 서울대학교 대학원 2021 국내석사

Nearly 30% of people worldwide does not have access to proper sanitation facilities due to reasons such as lack of water resources. Dry toilets have been introduced to overcome these challenges and urine diverting dry toilet (UDDT) is a type of a dry toilet that collects urine and feces separately. Human urine can be used as a good source of Nitrogen and phosphorous in agriculture but, prior to application urine should be treated. Many additives have been investigated that could stabilize urine but, in developing countries, usage of these materials is not economically feasible. As such, many researchers have investigated nature-based materials such as wood ash (WA) to use as a Magnesium source to promote struvite generation. However, when WA is used as an additive for phosphorous recovery, researchers have observed high heavy metal concentrations in the precipitate, rendering the precipitate unusable as a fertilizer. Banana is a cheap and widely available crop, and its peel ash (BPA) could be used as a good alternative. In this study, we investigated the effect of BPA in stabilizing fresh urine. When BPA was added in doses higher than 10 g/ 500 ml urine, the pH increased above 11 thus, urea hydrolysis was obstructed. Moreover, this research focused on investigating the effect of BPA on nitrogen and phosphorous recovery from synthetic wastewater and stored urine. Synthetic wastewater mixed with BPA doses above 10 g/ 500 ml showed no significant improvement in Nitrogen and Phosphate recovery. Nitrogen and Phosphate reduced significantly compared to the control sample due to struvite generation. This was validated by repeating the experiment on stored urine where, the precipitation demonstrated similar band pattern on XRD analysis as to that of struvite. Therefore, authors have concluded that the BPA can be used to recover nutrients in urine and a dosage of 10 g/ 500 ml of urine is optimum to achieve the highest struvite yield. Authors propose an innovative toilet design based on the findings of this research. 전 세계 인구의 30%가 물 공급을 제공받지 못하기 때문에 기본적인 화장실 시설을 이용하지 못하고 있다. UDDT(분뇨분리식 화장실)은 이러한 어려움을 극복하기 위해 도입된 화장실로 소변과 대변을 따로 모으는 건식 화장실의 일종이다. 사람의 소변은 농업에서 질소와 인의 좋은 공급원으로 사용될 수 있지만, 사용 전에 반드시 처리과정을 거처야 한다. 소변을 퇴비로 안정화 시킬수 있는 첨가제들이 개발되어왔지만, 개발도상국에서는 이러한 복잡한 과정과 정제된 물질의 활용이 현실적으로 어려운 환경이다. 이를 개선하기 위해 자연물을 활용한 개선책으로 나무재와 같은 물질이 연구 되었다. 나무재는 마그네슘 공급원으로 첨가되어 인산 회수를 위한 스트루바이트 생성에 활용할 수 있으나 침전물의 높은 중금속 농도로 인하여 비료로써 사용할 수 없다는 결론을 내렸다. 바나나는 값이 싸고 널리 사용가능한 자연 폐기물로 바나나 껍질 재(BPA)는 좋은 대안이 될 수 있다. 먼저 BPA는 갓 배출된 소변을 안정화 시키는 역할로 조사되었다. 소변 10 g/500 ml 이상 용량의 BPA를 첨가하면 산성도가 11 이상으로 상승하여 요소 가수분해가 억제되는것되었고 시간에 지남에 따라 소변내의 산성도가 10.5 미만으로 감소함으로 초기에 억제시킨 가수분해를 재개 시킬수 있다. 또한, 이 연구는 바나나 껍질 재가 인공 하수와 저장된 소변에서 스트루바이트 생성을 통해 질소 및 인산 회수에 미치는 영향조사에 초점을 맞췄다. BPA 농도를 10g / 500 ml 이상 초과하여 인공하수에 첨가하게 되면 10g / 500 ml 보다 질소 및 인산염 회수율에서 큰 개선을 보이지 않았다. 질소 및 인산염은 스트루바이트 생성으로 인해 대조군에 비해 현저히 감소되었다. 소변에 BPA를 넣고 반응시킨 침전물의 XRD 분석 결과는 표준 스트루바이트 패턴과 유사한 패턴을 보였다. 따라서, BPA가 소변의 영양분을 회수하는 데 사용될 수 있고 10 g/500 ml이 가장 최적이라고 결론 내렸다. 저자는 이 연구의 결과에 기초한 혁신적인 화장실 설계를 제안하였다.

Fate of nitrogen in urine separated toilet systems

Shervin, Hashemi 서울대학교 대학원 2015 국내석사

Present-day treatment of mixed waste water has several shortcomings: high amounts of resources, including drinking water, are consumed, valuable nutrients such as phosphorus, nitrogen or potassium are lost to the environment and micro pollutants are eliminated insufficiently. Source separation of urine, which contributes most of the nutrients to waste water, is a promising alternative. However, the nutrients in urine might not be available in a convenient form for fertilizers. Furthermore, urine contains micro pollutants such as synthetic hormones, pharmaceuticals and their metabolites. These substances are mainly excreted via urine and may be harmful to the ecosystems and human health. Today, many micro pollutants reach the aquatic environment, because their degradation in waste water treatment plants is poor. Urine treatment might be necessary to produce an adequate fertilizer, but it might also be a suitable method to prevent the pollution of the environment with micro pollutants. Developing a treatment method requires the knowledge about the concentration and behavior of the nitrogen compounds in urine. During separation, storage and transport, urine is subject to several spontaneous processes such as urea hydrolysis, which change the urine composition significantly. In this research fate of nitrogen in pure urine has been investigated. Also based on a wisdom learned from ancient Persian urine samples have been mixed with acetic acid and sodium bicarbonate and changes in fate of nitrogen and its effect in reducing odor of urine has been studied. 오늘날 대소변의 혼합된 처리는 여러 가지 문제점을 가지고 있다: 음용수를 포함한 많은 자원이 낭비되고 인, 질소, 칼륨과 같은 소중한 영양분들이 환경으로 배출되며, 미세오염물질이 비효율적으로 제거되고 있다. 소변의 대변으로부터의 분리는 효과적인 대안이다. 그러나, 소변이 가지고 있는 영양분들은 비료로 사용하기에 적합한 형태로 존재하지 않을 뿐만 아니라 소변은 인공호르몬, 의약물질, 대사산물과 같은 미세오염물질을 포함하고 있다. 이러한 물질들은 주로 소변을 통해 배출되며 생태계와 인간 건강에 해로울 수 있다. 이 물질들은 하수처리장에서 효율적으로 처리되지 못하고 있기 때문에, 오늘날 많은 미세 오염물질들이 그대로 수계로 배출되고 있다. 소변 처리는 적절한 비료를 생산하는 데에도 필요하며, 미세오염물질로 인한 환경오염을 막는 데에도 적절한 방법이 될 수 있다. 처리방법을 개발하는 것은 소변 내 질소화합물의 농도와 거동에 대한 지식을 필요로 한다. 소변의 분리, 저장과, 이동 동안 소변은 우레아 가수분해와 같이 소변의 구성을 크게 변화시키는 여러가지의 자동적인 공정을 거친다. 이 연구에서는 순수한 소변에서의 질소의 거동에 대해 알아보았다. 또한 과거 고대 페르시아로부터 배운 지식을 기반으로 하여, 소변 샘플들을 아세트산과 중탄산나트륨과 혼합하였을 때의 질소 거동의 변화와 소변의 악취를 제거하는 효과에 대해 연구하였다.

Advancing Membrane Technologies for Recovery of Phosphorus and Nitrogen from Human Urine

McCartney, Stephanie Nicole Columbia University ProQuest Dissertations & These 2022 해외박사(DDOD)

The existing linear economy approach to nutrient management has clear shortcomings including high expenditures for nutrient extraction and production of fertilizer as well as additional costs for nutrient removal at downstream waste water treatment plants (WWTPs) to prevent the pollution of aquatic environments. In a circular nutrient economy, phosphorus (P) and nitrogen (N) are removed from waste streams and captured as valuable fertilizer products in order to more sustainably reuse the resources in closed-loops and simultaneously protect receiving aquatic environments from harmful P and N emissions. The overarching aim of this thesis is to understand strategic approaches for nutrient recovery from wastewater and advance membrane technologies for P and N reclamation. The studies i.) approach nutrient recovery on a system-level to recognize optimal waste streams to target for P and N separation, ii.) advance membrane-based processes for nutrient recovery, and iii.) examine the economic viability of the nutrient recovery techniques. The thesis presents a thermodynamic and energy analysis of nutrient recovery from various waste streams of fresh and hydrolyzed urine, greywater, domestic wastewater, and secondary treated wastewater effluent. The analysis revealed comparative advantages in theoretical energy intensities for P and N recovery from nutrient-dense waste streams, such as fresh and hydrolyzed urine, compared to other more dilute sources. The thesis quantifies efficiencies required by separation techniques for nutrient reclamation to be competitive with the energy requirements of the prevailing industrial fertilizer production methods, i.e., phosphate mining and nitrogen fixation by the Haber-Bosch process. The dissertation examines and advances the performance of membrane-based processes for separation and recovery of P and N from diverted human urine. Donnan dialysis (DD), an ion-exchange membrane-based process, can capture and enrich orthophosphate, HxPO4(3-x)−, from source-separated urine. This work demonstrates the transport of Cl− driver ions down a concentration gradient, across an ion-exchange membrane to set up an electrochemical potential gradient that drives the transport of target HxPO4(3-x)− in the opposite direction, enabling P capture. Importantly, H2PO4− is transported against an orthophosphate concentration gradient, which achieves uphill transport of P. The thesis also provides a framework to better understand the impact of different ions in the water matrix on P recovery potential and kinetics. The thesis presents a novel operation of membrane distillation (MD)—isothermal membrane distillation with acidic collector (IMD-AC)—to selectively recover volatile ammonia, NH3, from hydrolyzed urine. The innovative isothermal and acidic collector features, respectively, suppressed undesired water permeation and enhanced ammonia vapor flux relative to conventional membrane distillation (CMD). The elimination of water flux in IMD-AC resulted in ≈95% savings in vaporization energy consumption relative to CMD. Critically, IMD-AC achieved uphill transport of ammoniacal nitrogen, i.e., transport against a concentration gradient, demonstrating the promising potential of the technique for N recovery. The dissertation proposes an integrated bipolar membrane electrodialysis (BPM-ED), DD, and IMD-AC system to drive the separation and recovery of orthophosphate and ammoniacal nitrogen from human urine. This work elucidates the role of pH and nutrient speciation (i.e., H2PO4− versus HPO42− and NH4+ versus NH3) on the performance of DD and IMD-AC. In the proposed configuration, BPM-ED generates acids and bases in situ to strategically control the pH of urine streams to benefit DD and IMD-AC performances. Strategic pH modification can enhance orthophosphate transport and selectivity in DD as well as ammonia transport and recovery potential in IMD-AC. Importantly, the analysis quantifies comparable specific energy consumptions of the proposed integrated membrane-based process to the existing approaches to P and N management. This thesis presents a preliminary economic assessment of onsite nutrient recovery employing DD and IMD-AC for respective P and N recovery from diverted urine. The analysis reveals opportunities to utilize widely-available waste chemical streams and recovered thermal energy to improve the economic viability of nutrient recovery. The largest capital expenditures are urine diversion toilets and additional piping for source-separation. Preliminary analysis demonstrates that employing urine diversion in public sanitation rooms, as opposed to private bathrooms, can reduce these capital expenditures. Furthermore, realizing savings from avoided costs for downstream nutrient removal at centralized wastewater treatment plants in addition to fertilizer revenue can enhance the economic viability of the approach. Overall, this dissertation critically informs nutrient recovery approaches and advances membrane-based processes for P and N reclamation to facilitate a paradigm shift from an inefficient linear nutrient economy to a sustainable circular nutrient economy. The work reveals opportunities to minimize energy intensity for nutrient separation, advance the performance of membrane-based techniques for selective and energy-efficient nutrient recovery from urine, and enhance the cost-competitiveness of nutrient reclamation. The findings of this work support nutrient recovery efforts and provide important insights that can be applied to other separation and resource recovery endeavors.

질량분석법과 결합된 크로마토그래피 기법을 이용한 소변 중 칸나비노이드 대사체 분석

심영은 경성대학교 일반대학원 2023 국내박사

소변 칸나비노이드 대사체인 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-glucuronide (Δ9-THCCOOH-glu), and 7-carboxy-cannabidiol (7-COOH-CBD)을 분석하기 위해 질량분석법과 결합된 크로마토그래피 기법을 개발하였습니다. 정성 주입구 유도체화 –기체크로마토그래피 질량분석법 (IPD–GC–MS)은 소변 중 Δ9-THCCOOH를 측정하기 위하여 개발되었습니다. 소변 중 매트릭스 효과를 줄이고 Δ9-THCCOOH을 효과적으로 추출하기 위하여 고속원심분리 (high-speed centrifugation) 방법을 도입하였습니다. 또한 유도체화 과정의 시간을 줄이기 위해 N,O-bis(trimethylsilyl)-trifluoroacetamide containing 1% trimethylchlorosilane (BSTFA+1% TMCS)을 함유한 아세트산을 이용하여 주입구 유도체화법을 개발하였습니다. 정성 IPD–GC–MS 방법은 선택성, 검출한계 (LOD), 재현성, 민감도, 특이성, 정밀도 및 정확도 항목으로 검증하였습니다. 개발된 분석법의 적용 가능성은 공-소변 (blank urine)에 표준물질을 첨가하여 제조된 시료와 대마초 남용자로 얻은 소변 (n = 221)을 분석하여 평가하였습니다. 그 결과, 개발된 정성 IPD–GC–MS 방법의 유용성을 확인할 수 있었습니다. 소변 중 Δ8-THCCOOH, Δ9-THCCOOH, Δ9-THCCOOH-glu, 및 7-COOH-CBD을 검출하기 위한 자동화 온라인 마이크로 고체상–액체크로마토그래피 이중 질량분석법 (μSPE–LC–MS/MS)을 개발하고 검증하였습니다. 자동화 온라인 μSPE를 위한 장치를 LC–MS/MS에 결합한 후, 소변 매트릭스를 효과적으로 줄이고 4종의 분석 대상물질의 효과적인 추출을 위해 ITSP (instrument top sample preparation) 카트리지가 도입하였습니다. 크로마토그래피 조건 (컬럼, 컬럼 온도, 이동상, 유속)을 최적화하여 이성질체 관계인 Δ8-THCCOOH과 Δ9-THCCOOH의 화합물을 완벽하게 분리하였습니다. 개발된 분석법은 산업을 위한 생물학적 분석방법 검증 지침 (Bioanalytical Method Validation Guidance for Industry, 2018)에 따라 선택성, 검출한계 (LOD), 최저 정량한계 (LLOQ), 직선성, 정확성, 정밀성, 희석 무결성, 회수율 (RE), 기질효과 (ME) 및 안정성 항목으로 검증하였습니다. 소변 중 4종의 대마 대사체 분석이 가능한 자동화 온라인 소변 분석법은 대마 남용 의심자 소변 78개를 효과적으로 분석할 수 있었습니다. Analytical methods for chromatography technique coupled with mass spectrometry has been developed for the determination of cannabinoid metabolites, such as 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-glucuronide (Δ9-THCCOOH-glu), and 7-carboxy-cannabidiol (7-COOH-CBD). The qualitative injection-port derivatization (IPD)–gas chromatography–mass spectrometry (GC–MS) method was developed for the detection of Δ9-THCCOOH in human urine. For purification of the urine extract prior to instrumental analysis, high-speed centrifugation was used to minimize interference. In addition, an injection-port derivatization method using ethyl acetate and N,O-bis(trimethylsilyl)- trifluoroacetamide containing 1% trimethylchlorosilane (BSTFA+1% TMCS) was employed to reduce the time required for derivatization, and an aliquot of the final solution was injected into the GC–MS. The method was validated by selectivity, LOD, repeatability, sensitivity, specificity, precision, and accuracy. The applicability of the developed method was examined by analyzing spiked urine and forensic urine samples obtained from suspected cannabis abusers (n = 221). An automated online micro-solid-phase extraction (μSPE)–liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the detection of Δ8-THCCOOH, Δ9-THCCOOH, Δ9-THCCOOH-glu, and 7-COOH-CBD in urine. An instrument top sample preparation (ITSP) cartridge was introduced to increase the sensitivity toward analytes and decrease the matrix effect of the urine. The chromatographic separation of Δ8-THCCOOH and Δ9-THCCOOH was achieved through adjustment of LC parameters. The analytical method was validated for selectivity, the limit of detection (LOD), the lower limit of quantification (LLOQ), linearity, accuracy, precision, dilution integrity, recovery (RE), matrix effect (ME), and stability according to the Bioanalytical Method Validation Guidance for Industry. The developed method was successfully applied to the analysis of 78 urine samples.

요중 백혈구 에스터라제의 활성을 측정하기 위한 진단용 기질의 합성에 관한 연구

박정오 檀國大學校 大學院 1997 국내박사

백혈구(leukocyte)는 인체가 세균이나 진균, 바이러스 등에 의해 감염이 되면 그 숫자가 증가하게 된다. 특히, 요 중에서 백혈구가 검출되면 신장을 비롯한 비뇨기계와 생식기계의 감염을 의심하게 되므로 이 계통의 질병을 진단하고 치료 및 예후를 판정하는 데 중요한 지표가 되고 있다. 요 중에 포함되어 있는 백혈구를 검출하는 방법으로 원심 분리하지 않은 요와 원심분리 한 요 침전물을 현미경으로 직접 검경하여 백혈 구 수를 세는 방법이 이용되고 있으나 자가용해와 다른 세포성분과의 감별 등에 고도의 지식이 필요하며, 알칼리성 요에서는 반감기가 60분 정도이므로 그 이후에는 백혈구를 검출할 수 없어 위음성을 나타 낼 우려가 있다. 또한, 현미경과 원심 분리기 등 별도의 장치가 필요하며, 시료의 전처리 등으로 인하여 신속성이 결여되어 있다. 이와 같은 단점을 보완하기 위하여 백혈구가 요 중에 용해되어 있거나 용해되어 있지 않더라도 백혈구 에스터라제에 특이성이 있는 기질을 사용하므로서 신속, 간편하며 감도와 정확성을 높일 수 있고, 요 중의 다른 성분에대한 영향이 비교적 적은 기질을 개발하는 데 중점을 두었다. 따라서, 요 중의 백혈구 측정에 미치는 영향을 최소와 할 수 있는 기질을 개발하여 합성된 발색성 기질과 형광성 기질이 에스터라제에 의해 가수분해되어 유색의 산과 알코올이 유리되면 디아조늄염과 아조 짝지움 반응을 일으켜 착색 화합물 또는 형광색소가 형성된다. 이때, 발색되는 정도는 요중 백혈구 에스터라제의 활성도에 비례하므로 요 중에 존재하는 백혈구 수를 간접적으로 측정할 수가 있다. 본 연구에서는 백혈구 에스터라제의 활성을 이용하여 요 중에 백혈구가 존재할 경우 착색 반응을 일으키는 백혈구 측정용 시험지를 개발하여 보고 하는 바이다. 우선, 경제성이 있는 에스테르 기질을 합성하기 위하여 펜틸 알코롤 �薦聆臼�삼 플루오르화 붕소 디에틸 에테레이트를 산 촉매로 사용하는 친핵성치환 반응을 이용하였다. 즉,N,S 원자를 포함하고 있는 2-pyrazine,3-thiophene,8-quinoline의 카르복실산과 반응시켜 에스테르 기질을 합성한 후, 기존의 N-보호화 된 아미노산을 함유하고 있는 기질과 비교하였던 바, 3-thiophene과 8-quinoline 에스테르 기질은 감도와 안정성이 현저하게 낮았다. 또한, 2개의 질소 원자를 함유하고 있는 2-pyrazine 에스테르의 경우에는 상대적으로 높은 감도를 나타냈으나 기존에 사용하고 있는 아미노산 에스테르 기질보다는 감도와 정확성이 떨어지는 것을 관찰하였다. 한편, 형광성 기질로 플루오레세인 에스테르 유도체를 이용하였을 경우에는 감도가 높아 반응 즉시 육안적인 식별만으로도 뚜렷하게 구분할 수 있었으나, 요중 백혈구수의 양성 반응 단계를 등급별로 평가할 수 있는 특별히 고안된 측정 장치의 개발이 요구되었다. 따라서, 요 중의 백혈구 에스터라제는 N-보호화 된 아미노산 혹은 펩티드로 유도된 기질에 특이성을 보인다고 추론할 수 있었고, 이를 보완하기 위하여 티아졸 유도체가 인돌이나 피롤과 같이 에스터라제에 의해 가수분해 되면 헤테로 고리 내에 풍부한 전자 밀도가 형성되어 강한 친핵성을 나타낸다는 점과 에스터라제가 N-보호화 된 아미노산 유도체 기질에 특이성이 있다는 것을 기초로 하여 5원 고리에 황과 질소 원자가 결합되어 있는 티아졸 유도체에 N-보호와 된 아미노산을 결합시켜 에스테르 기질을 합성 하였다. 합성된 기질을 이용하여 백혈구 검출용 시험지를 제조한 후 요준의 백혈구를 측정하였던 바, 감도, 안정성, 반응시간 및 정확도가 기존에 시판되고 있는 요중 백혈구 검출용 진단 시약을 이용한 시험지법과 동일하거나 더 나은 효능을 나타냈으며 합성 과정이 까다로운 종래의 기술과는 달리 합성의 전과정이 단순하여 일반적인 방법으로 짧은 시간내에 최종 생성물에서 순수한 에스테르 기질을 분리하여 수득율을 향상 시킬 수 있었다. 또한, 반응 색상이 안정하여 표준 비색표에 의한 육안적인 감별은 물론, 요화학 자동 분석 기에서의 응용성이 좋았다. 본 연구를 통하여 요중에 존재하는 백혈구 측정용 시험지법을 개발하게 되었다. 이를 계기로 대부분이 수입에만 의존하고 있는 임상 진단용 시약에 대한 국산화에 이바지 할 것을 기대하면서, 보다 더 민감하고 안정되며 반응 단계가 뚜렷하게 구분 될 수 있는 기질과 진단용 시약의 개발에 더욱 더 많은 연구가 진행되어야 할 것으로 사료된다. A number of leukocyte are increased when one is infected by a micro-organism. Especially, the detection of leukocyte levels can be an inportant indication of such medical informations as kideny or urogenital tract infection or other dysfunction. Among the analytical techniques of leukocyte analysis, the microscopic leukocyte analysis of uncentrifuged urine and urine sediment has veen used requiring a high knowledge in making distinction between self-lysed cells and other urinary components such as epithelial cells and salt particles. Moreover, in urine of abnormally high pH, the half life of leukocyte can be as low as 60 minutes. The lysed cells beyond the detection of visual examination result in false negative. In addidion, the equipment such as a microscope and centrifuge are required and a lacking of rapodness appears because of the pretreatment of samples. To make up the disadvantage, the synthesis of a rapod, facile, sensitive, and accurate substrate for detecting leukocyte esterase activity in urine was primarily emphasized. Therefore, the synthesized chromogenic and fluorogenic esters which, when hydrolyzed by esterase in urine, can be used as substrates for determining esterolytic enzymes, produce a colored alcoholic product which is in turn coupled with diazonium salt to form the corresponding azo dye. As the extension of color formed is proportion to the enzymatic activity in urine sample, the munber of leukcyte can be measured. In thes stydy, the economic and technical leukocyte esterase substrates were synthesized by esterification of heterocyclic carboxylic acids such as 2-pyrazine, 3-thiophene, and 8-quinoline using a boron trifluoride diethyl etherate-alcohol reagent in the presence of pentyl alcohol because several heterocyclic ring systtems are susceptible to hydrolytic degradation in the presence of strong acids and bases and were tested for their stability and sensitivity compared with the presented N-blocked amino acid or peptide esterase substrate. The stability and sensitivity of 2-pyrazine esterase substrate. The stability and sensitivity of 2-pyrazine esterase substrate, which has two nitrogen atoms, was higher than that of 3-thiophene and 8-quinoline derivatives but still lower than that of N-blocked amino acid esterase substrate. Therefore, it is suggested that the N-blocked amino acid esterase substrate has the specificity for the leukocyte esterase in urine. Based on the suggestion that when thiazole derivatives were hydrolyzed bu esterase just like as indole and pyrrole derivatives, the Nblocked amino acid derivatives have the specificity because of strong nucleophilic property from high electron density in heterocyclic ring. The OH group of thiazole derivatives was reacted with N-tosyl alaninyl chloride to give N-blocked amino acid thiazole ester. This substrate showed higher sensitivity, stability, reaction time, and accuracy then with the presented N-blocked amino acid esterase substrate and also had a berrer visual determination. The process of synthetic reaction ws simple and produced a good yield in comparion with the presented difficult technique. The N-blocked amino acid dervative fluoresceine ester as fluorogenic eaterase substrate was synthesized. This characteristic substrate has a fast reaction time in addition to a visual determination but the positive reaction steps could not slassified. Especially it required a special instrument to detect leukocyte esterase activity. Through thes research, the quick and facile method for detecting leukocyte levels in urine was developed by synthesizing an dvanced chromogenic substrate for leukocyte substrate activity. It will contributr to homemade diagnostic agents which currently depends on import. The further research should be maintained for developing more sensitive and stable substrate in addition to the distinguished reactioon steps.

Phthalates and alternative plasticizers exposure characterization and associated reproductive health outcomes in women of reproductive age

이고운 서울대학교 대학원 2020 국내박사

Exposure to endocrine disrupting chemicals (EDCs) is important in women of reproductive age in terms of related adverse health effects. Personal care products (PCPs) is one of the common sources of EDCs. Considering the frequent and excessive use of PCPs by women, EDCs exposer through the use of PCPs is of great concern to the female population. The prenatal period could be the most sensitive period since exposure to EDCs could affect fetal development and the health of later life stage. It was important to evaluate and manage EDCs exposure during pregnancy. Phthalates are known as EDCs displaying anti-androgenic and weak estrogenic properties. Phthalates are often used as plasticizers. Due to increasing health concerns of phthalates, their uses in many applications were regulated recently worldwide. Subsequently, alternative plasticizers have been used in increasing amounts. The alternative plasticizers with a similar chemical structure of phthalates might have endocrine disruption function. Biomonitoring studies for the alternative plasticizers, however, were mostly limited to several European countries. A few studies have reported on the related health effects of alternative plasticizers. The present study was conducted to assess related reproductive health effects and to characterize exposure of plasticizers in women of reproductive age. Uterine fibroids were selected as reproductive health effects of exposure to phthalates and their alternatives. For the purpose, we recruited premenopausal women in Korea, and the severe cases (n = 32) and controls (n = 79) were subsequently selected based on the results of gynecologic ultrasonography for the diagnosis of uterine fibroids, and measured for metabolites of organophosphate ester (OPEs), alternative plasticizers, and phthalates in urine. To evaluate the characteristics of plasticizer exposure in the most vulnerable populations, pregnant women were recruited from Korea (n = 81) and Thailand (n = 102). Twenty- four metabolites of 15 phthalates were measured in urine samples collected once each trimester, i.e., three times during pregnancy. To understand the changes in urine characteristics during pregnancy, concentrations of urinary correction factors, such as creatinine, specific gravity (SG), and osmolality, were measured in urine samples of the participants. The first study (Chapter 2) suggested the importance of exposure to alternative plasticizers including phthalates in female reproductive health. Among alternative plasticizers, metabolites of di-isononyl phthalate (DINP) and di(2-propylheptyl) phthalate (DPrHpP) were detected in >75% of the urine samples. Among OPEs metabolites, diphenyl phosphate (DPHP), 2-ethylhexyl phenyl phosphate (EHPHP), and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP) were detected in >80% of the subjects. The concentrations of mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), the sum of five di(2-ethylhexyl) phthalate metabolites (∑5DEHP), and mono(4-methyl-7-hydroxyoctyl) phthalate (OH-MINP) were significantly higher in patients with uterine fibroids than in control participants. We found for the first time that several alternative plasticizers, such as di(2-ethylhexyl) terephthalate (DEHTP), DPrHpP, DINCH, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-butoxyethyl) phosphate (TBOEP) were associated with increased risks of uterine fibroids among Korean women. While the mechanisms of those plasticizers leading to uterine fibroids are not well understood, sex endocrine disruption by these compounds may provide one potential explanation. In the second study (Chapter 3), we described the exposure profile of gestational phthalates and alternative plasticizers and identified important plasticizers. While the levels of several phthalate metabolites were significantly different by trimester among Korean pregnant women, they were relatively stable across the three trimesters of pregnancy among Thai pregnant women. Urinary metabolites of diethyl phthalate (DEP) and dioctyl phthalate (DnOP) were two to three times higher in Thai pregnant women than those in Korean pregnant women. This observation may be linked to the more frequent use of cosmetics which contain DEP and DnOP in Thai than in Korean women. The detection frequencies of a metabolite of DINCH were 67.4% and 44.9% among the Korean and Thai pregnant women, respectively. According to risk assessment, Korean and Thai pregnant women were considered at high risk of 11.9% and 5.3%, respectively, due to phthalate exposure, particularly DEHP, DnBP and DiBP. These observations indicated that the plasticizer exposure profile was clearly different by country and trimester. In addition, DINCH has been used by pregnant women in Korean and Thai. The third study (Chapter 4) described the variations of urine characteristics in pregnancy. Three urinary correction factors were measured in urine samples collected in a pregnant female population of the second study. Among Korean pregnant women, urinary concentrations of creatinine and osmolality were significantly lowered at T3 than T1 suggesting more diluted urine as later stages of pregnancy. Those factors, however, were stable across the trimester of gestation in Thai pregnant women. Nationality or lifestyle may affect the changes in urinary dilution status during pregnancy. The correlations between unadjusted and adjusted concentrations during pregnancy were less than those of each adjusted phthalate concentrations. These results suggest that the variations of urinary correction factors may make the uncertainty propagation in exposure assessment of chemicals for pregnant women with urine samples. It is necessary to understand the changes in urinary correction factors for dilution by trimesters and nationality for a reduction in exposure misclassification of pregnant women. In addition, it needs to be careful at understanding the exposure biomarker during pregnancy in terms of the changes in urine volume or a series of characteristics related to the absorption, distribution, metabolism, and excretion (ADME) of chemicals in the body. The series of studies demonstrated important health effects of plasticizers and exposure characterization and in women of reproductive age. We determined the important emerging plasticizers associated with uterine fibroids. In addition, exposure profiles of plasticizers varied across the trimester of pregnancy. Variations of urine dilution during pregnancy due to physiological changes varied by country. These findings suggest that variations of exposure and related factors should be considered during study design and interpretation of results in biomonitoring studies of pregnant women. Considering the increasing levels of occurrences of alternative plasticizers in both environment and biospecimen, continuous exposure and risk assessment for other alternative plasticizers are warranted. Since the sample size of the present study is small, our observations need to be confirmed in larger cohorts or through experimental study. The present study is expected to contribute to improving the exposure assessment and management of EDCs in the vulnerable population. 가임기 여성의 내분비계교란물질(endocrine disrupting chemicals, EDCs)의 노출은 노출과 건강 영향 측면에서 중요하다. 개인생활용품 사용을 통해 노출되는 EDCs의 경우, 여성은 남성보다 개인생활용품을 더 자주, 그리고 더 많은 양을 사용하므로 그 노출량이 여성에서 훨씬 크다. EDCs 노출은 생식에 직접적인 영향을 줄 수도 있다. 특히 가장 민감한 시기라고 할 수 있는 임신기의 EDCs 노출은 태아의 초기 발달과 태어난 이후 평생에 걸쳐 건강에 영향을 미칠 수 있다. 그러므로 가임기 여성을 대상으로, 특히 노출과 건강 영향에 민감한 시기인 임신중 EDCs의 노출과 영향을 평가하고 관리하는 것은 중요하다. 가소제로 사용되는 프탈레이트는 항안드로겐성 및 약한 에스트로겐성을 보이는 대표적인EDCs이며, 내분비계 교란을 통해 사람의 건강에 영향을 미친다. 그 독성 때문에 여러 국가에서는 프탈레이트의 사용을 제한하고 있으며, 이로 인해 대체 가소제의 사용이 점점 증가하고 있다. 프탈레이트의 내분비교란성을 고려했을 때, 구조적으로 유사한 대체 가소제 역시 내분비계 교란을 일으킬 가능성이 있지만, 관련된 건강 영향 연구는 매우 부족한 실정이다. 특히나 대체 가소제의 바이오모니터링 연구는 대부분 유럽국가를 대상으로만 수행되었으며, 아시아 지역에서의 보고는 미미하다. 본 연구는 가임기 여성에서 프탈레이트 및 대체 물질의 생식건강 영향을 평가하고, 특히 화학물질 노출에 민감한 시기인 임신기의 가소제 노출 특성을 파악하고 평가하기 위해 수행되었다. 프탈레이트 및 대체물질 노출의 여성 생식 건강 측면에서 중요성을 파악하기 위해, 자궁근종 환자 대조군 연구를 수행하였다. 이를 위해 가임기 여성을 모집하여 자궁근종 진단을 위한 부인과 초음파 검사를 수행하였다. 검사 결과를 바탕으로 32명의 환자와 79명의 대조군을 선택하고 소변시료에서 프탈레이트와 대체 가소제, 유기인계난연제의 대사체 수준을 측정하였다. 가장 민감한 시기인 임신기의 가소제 노출 특성을 파악하기 위해, 한국과 태국에서 각 81명과102명의 임산부를 모집하였다. 임신 분기별 각1회, 즉 임신중 총 3회 수집된 소변시료에서 총 15종 프탈레이트의 24종의 대사체를 측정하였다. 임신기 소변 시료를 이용한 노출평가에서, 임신중 소변의 특성 변화를 이해하기 위해 소변보정지표인 크레아티닌, 비중, 삼투질농도를 측정하여 그 변이를 기술하였다. 전체 연구는 세 부분으로 나누어 다음과 같이 수행하였다. 첫번째 연구는 여성 생식 건강 측면에서 프탈레이트를 포함한 신규 가소제류의 노출의 중요성을 자궁근종 사례연구로 확인하기 위하여 수행되었다. 대체 가소제인, i-isononyl phthalate (DINP) 와 di(2-propylheptyl) phthalate (DPrHpP) 대사체가 소변중 75% 이상 검출되었고, 가소제로도 사용되는 유기인계난연제의 대사체중에는 diphenyl phosphate (DPHP), 2-ethylhexyl phenyl phosphate (EHPHP), 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP)가 80% 이상 검출된것으로 보아, 우리나라 가임기 여성에서 대체가소제가 널리 사용되고 있음을 의미한다. 대조군보다 환자군에서 DEHP 대사체 농도의 총합이 유의하게 높았고, (2-ethylhexyl) terephthalate (DEHTP), DPrHpP, DINCH, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(2-butoxyethyl) phosphate (TBOEP)의 노출이 자궁근종에 대한 오즈비를 증가시키는 것으로 나타났다. 이러한 연관성에 대한 생물학적인 기전으로는DEHTP, DINCH, TDCIPP, TPOEP물질의estrogenic한 성질이 자궁근종을 일으키는데 기여했을 것으로 생각된다. 이 연구는 대체 가소제류와 자궁근종간의 연관성을 보고한 첫 연구이다. 두번째 연구는 임신기 프탈레이트와 대체 가소제의 노출 양상을 기술하고 위해도가 큰 주요한 물질을 찾기 위해 수행되었다. 한국 임산부에서 프탈레이트 대사체 중 몇 종의 농도는 임신 시기에 따라 큰 변화를 보였지만, 상대적으로 태국 임산부에서는 그 변이가 작았다. 태국 임산부의 diethyl phthalate (DEP)와 dioctyl phthalate (DnOP) 대사체의 농도가 한국 임산부에서보다 2 – 3배 높았으며, 노출원을 고려했을 때, 태국 임산부의 화장품사용과 관련이 있을 것으로 보인다. 대체 가소제인 di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH) 대사체 검출빈도는 한국에서 67.4%, 태국에서 44.9%였다. 프탈레이트 및 대체 가소제의 위해성평가 결과, 한국에서는 11.9%, 태국에서는 5.3%의 산모가 위해 지수 1을 초과하였으며, 두 국가 모두에서 di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), di-2-isobutyl phthalate (DiBP)의 노출이 주요 요인인 것으로 나타났다. 이러한 결과는 가소제 노출 특성은 임신기간에 따라, 또한 국가에 따라 분명한 차이가 있음을 나타낸다. 세번째 연구에서는 임신기 소변의 특성 변화를 이해하고, 임신으로 인한 생리학적 변화가 노출평가에 갖는 중요성을 강조하였다. 두번째 연구에서 수집한 임산부 소변시료에서 소변 묽기 보정 지표 세가지를 측정하여 그 변이를 기술하였다. 한국 산모에서는 크레아티닌과 삼투질 농도가 임신 전기보다 후기에서 낮아졌는데, 이는 임신 후반기로 갈수록 소변이 더 묽어진다는 것을 의미한다. 그러나 태국 임산부에서는 임신 3분기 동안 소변 묽기보정 인자의 수준이 일정했다. 캐나다, 일본 산모연구 결과와 함께 고려했을 때, 임신중 소변 묽기보정 인자의 변화가 국가 또는 생활 방식에 따라 다르게 나타날 수 있음을 의미한다. 임신중 프탈레이트 노출량은 소변 묽기 보정을 한 농도간 높은 연관성을 보이며, 이에 비해 묽기 보정 전후간 연관성은 낮게 나타났다. 소변 보정지표의 선택에 따라 임신3분기 프탈레이트 농도 변화 양상의 통계적 유의성에 차이를 나타냈다. 이 결과는, 임신중 체내 생리학적 변화로 인한 소변 보정 지표의 변화가 임산부의 화학물질 노출평가의 불확실성을 크게 만들 수 있음을 의미한다. 그러므로 임신기 소변중 오염물질의 측정을 통한 노출평가에서 임신 분기별, 국가별로 소변 묽기보정 인자의 변화 양상을 이해해야 한다. 또한 임신중 소변량의 변화나 체내 화학물질의 ‘흡수, 분배, 대사, 배출’과 관련된 일련의 특성 변화를 함께 고려하여 임신중 노출 바이오마커를 해석해야 할 것이다. 프탈레이트를 포함한 가소제를 중심으로, 가임기 여성 집단에서의 EDCs 노출과 관련된 여성 생식 건강 영향과 노출 특성을 관찰한 세 단면 연구를 통해, 대표적인 여성 생식 질환인 자궁근종과 연관성을 보이는 신규 가소제 물질을 파악하였다. 또한, 가장 민감한 임신기의 프탈레이트류 노출 양상이 임신 분기에 따라 다양하며, 임신중 소변 묽기가 변하는 양상이 국가별로 다양하다는 것을 확인하였다. 이 결과는 임산부의 소변 샘플을 사용한 바이오모니터링 연구에서, 연구 설계 및 결과 해석 과정에서 노출 및 관련 인자의 변이를 고려해야 함을 시사한다. 또한, 대체 가소제의 증가하는 사용 추세로 미루어, 향후 다양한 신규 가소제에 대한 지속적인 노출 및 건강 영향 평가가 필요할 것이다. 본 연구 결과는 적은 수의 표본을 통해 도출되었기 때문에, 더 큰 표본수의 인구집단을 대상으로 하는 후속 연구 혹은 실험연구를 통한 보완되어야 할 것이다. 본 연구는 민감인구에서 EDCs의 노출을 정확히 평가하고, 관리하는데 기여할 수 있을 것이다. 이를 통해 사회 전반적으로 EDCs 노출을 줄여나가는 노력에 도움이 될 수 있을 것으로 기대한다.

Gas chromatography-mass spectrometry를 이용한 human urine에서의 내인성 steroid hormone의 분석에 관한 연구

장현경 建國大學校 大學院 1991 국내석사

The seventeen endogencous steroids from human urine were simultaneously analyzed by selected ion monitoring method of GC/MS. Urine samples were collected for twenty-four hours from sixty-eight of normal males, thirty-four normal females and eight of normal children. Urinary steroids were extracted by using XAD-2 resin, hydrolyzed with β-glucuronidase/arylsulfatase from Helix pormatia and quantitatively derivatized by N-methylsilylheptafluorobutyramide/trimethylsilylimidazole/trimethylchlorosilane mixture in order to detect on the GC/MS. The observed concentrations of seventeen endogeneous steroids in human urine were in the range of 0.01~300 ㎍/ml. The ratio of their precursor and product metabolite of endogeneous steroids were shown in the range of 0.05~23.5. This study will be expanded to determine normal reference values for steroid in urine for Korean. Once normal reference values are obtained, the comparison data of the quantitative results of endogeneous steroids in human urine will be used as a part of diagnostic tool for endocrine disease.

Advancing the Implementation and Adoption of Urine Diversion Systems in Commercial and Institutional Buildings in the United States: A Focus on Control of Urea Hydrolysis

Saetta, Daniella ProQuest Dissertations & Theses Arizona State Univ 2021 해외박사(DDOD)

This dissertation focused on the implementation of urine diversion systems in commercial and institutional buildings in the United States with a focus on control of the urea hydrolysis reaction. Urine diversion is the process by which urine is separately collected at the source in order to realize system benefits, including water conservation, nutrient recovery, and pharmaceutical removal. Urine diversion systems depend greatly on the functionality of nonwater urinals and urine diverting toilets, which are needed to collect undiluted urine. However, the urea hydrolysis reaction creates conditions that lead to precipitation in the fixtures due to the increase in pH from 6 to 9 as ammonia and bicarbonate are produced.Chapter 2 and Chapter 3 describes the creation and use of a cyber-physical system (CPS) to monitor and control urea hydrolysis in the urinal testbed. Two control logics were used to control urea hydrolysis in realistic restroom conditions. In the experiments, acid was added to inhibit urea hydrolysis during periods of high and low building occupancy. These results were able to show that acid should be added based on the restroom use in order to efficiently inhibit urea hydrolysis.Chapter 4 advanced the results from Chapter 3 by testing the acid addition control logics in a real restroom with the urinal-on-wheels. The results showed that adding acid during periods of high building occupancy equated to the least amount of acid added and allowed for urea hydrolysis inhibition. This study also analyzed the bacterial communities of the collected urine and found that acid addition changed the structure of the bacterial communities.Chapter 5 showed an example of the capabilities of a CPS when implemented in CI buildings. The study used data mining methods to predict chlorine residuals in premise plumbing in a CI green building. The results showed that advance modeling methods were able to model the system better than traditional methods. These results show that CPS technology can be used to illuminate systems and can provide information needed to understand conditions within CI buildings.

Simultaneously determination of 14 hypoglycemic drugs in human urine by liquid chromatography - tandem mass spectrometry

Truong, Quoc-Ky Kangwon National University 2018 국내박사

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of 14 oral hypoglycemic drugs in human urine. For the first time, metformin and phenformin which are highly polar compounds were simultaneously separated and quantified with other less polar hypoglycemic drugs. Metoprolol was used as internal standard (IS). Oral hypoglycemic drugs were chromatographed on a Hilic column (50 x 3.0 mm i.d., 3 µm) combined with C18 guard column (4.0 x 3.0 mm i.d.) using a 30:70 (v/v) mixture of 1 mM ammonium formate pH 5.0 (mobile phase A) and acetonitrile (mobile phase B) as mobile phase at a flow rate of 0.35 mL/min, column temperature was at 350C. The gradient program started from 0% mobile phase B, linearly increased to 70% B from 0.2 to 10.0 min and then decreased to the start condition (0% mobile phase B) in 1 min. Mobile phase B was held at 0% for 9.0 min (from 11.0 min to 20.0 min). The total run time was 20.0 min. API-3200 LC-MS/MS in multiple reaction monitoring (MRM) mode was used for detection. Mass spectrometers and production ion mass spectrum of all standards were operated in the positive ion detection mode with an ESI source for mass analysis and detection. Parameters of MS were optimized with analyst software (version 1.4.2) to process the data with the guidelines suggested by the manufacturer (AB Sciex, 2013). The method developed was validated in terms of system suitability, specification, sensitivity (the detection limit LOD, the quantification limit LOQ), linearity, carryover, qualitative matrix effect precision and accuracy, robustness, stability according to ICH (Q2 2005) and bioanalytical method validation FDA guidance. The calibration curves were linear over the concentration range of 2-300 ng/ml for sitagliptin, rosiglitazone, pioglitazone, repaglinide, phenformin and mitiglinide, 5-400 ng/ml for glipizide, gliclazide, nateglinide, glibenclamide, glimepiride, metformin and 20-500 ng/ml for tolbutamide, chlorpropamide with r2 = 0.99. The quantification limit LOQ was 2 ng/ml for sitagliptin, rosiglitazone, pioglitazone, repaglinide, phenformin and mitiglinide, 5 ng/ml for glipizide, gliclazide, nateglinide, glibenclamide, glimepiride, metformin and 20 ng/ml for tolbutamide, chlorpropamide. Criteria for acceptability of the data included intraday and interday precision and accuracy at LOQ concentration were below 20%; at medium, low and high concentration were below 15%. The method was precise and sensitive enough for the simultaneous determination of 14 hypoglycemic drugs in urine. . Recovery and matrix effect of the drugs ranged between 80% and 99%: metformin (82% - 93%), chlorpropamide (82% - 96%), phenformin (80% - 84%), sitagliptin (81% - 95%), tolbutamide (80% - 86%), gliclazide (80% - 82%), glipizide (86% - 99%), mitiglinide (81% - 89%), nateglinide (80% - 81%), rosiglitazone (81% - 90%), pioglitazone (80% - 87%), glibenclamide (80% - 85%), glimepiride (80% - 82%) and repaglinide (82% - 93%). The criteria for acceptability calculated for the concentration was not more than 15%. Stability of all compounds at each level (LOQ, low, medium and high) in short-term and long-term had the maximum relative error (%) of 14.34% which was within the criteria (±15% of the nominal concentration). Selectivity, system suitability, carry over and robustness of the validation method were within the acceptable criteria. This analytical method was successfully applied to a human urine sample and metformin and nateglinide could be effectively detected and quantitated after two days of the administration of the drugs.