KR101426529B1 - Evaluation of Seasonal Variations of Cholesterols in Hair Fragments - Google Patents
Evaluation of Seasonal Variations of Cholesterols in Hair Fragments Download PDFInfo
- Publication number
- KR101426529B1 KR101426529B1 KR1020120121162A KR20120121162A KR101426529B1 KR 101426529 B1 KR101426529 B1 KR 101426529B1 KR 1020120121162 A KR1020120121162 A KR 1020120121162A KR 20120121162 A KR20120121162 A KR 20120121162A KR 101426529 B1 KR101426529 B1 KR 101426529B1
- Authority
- KR
- South Korea
- Prior art keywords
- cholesterol
- hair
- present
- concentration
- compounds
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01021—7-Dehydrocholesterol reductase (1.3.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01072—DELTA24-sterol reductase (1.3.1.72)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13017—Cholesterol 7-alpha-monooxygenase (1.14.13.17)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
본 발명은 머리카락 내 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성에 대한 계절별 변화를 측정하는 방법 및 이를 활용한 콜레스테롤 관련 질환의 유무, 발병 가능성 또는 호전의 정도를 예측하는 방법에 관한 것이다. 본 발명에 따르면, 한 올의 머리카락만으로도 인체 내 콜레스테롤 화합물 농도의 계절별 변화를 매우 정확히 측정하여 기존의 주기적인 혈액 검사보다 안전하고 손쉬운 방법으로 측정할 수 있다. 또한, 본 발명은 기존의 혈액 시료 채취의 어려움을 보완하고, 실질적으로 건강검진에 적용하여 보다 쉽게 콜레스테롤 대사 변화와 관련되는 질환들을 예측할 수 있는 장점을 갖는다.The present invention relates to a method for measuring seasonal changes in cholesterol compound concentration in hair or metabolic enzyme activity of cholesterol compounds and a method for predicting the presence, occurrence or improvement degree of cholesterol-related diseases using the same. According to the present invention, seasonal changes of the cholesterol compound concentration in the human body can be measured very accurately with a single-stranded hair, so that it can be measured more safely and easily than conventional periodic blood tests. In addition, the present invention has an advantage of being able to predict diseases associated with cholesterol metabolism change more easily by supplementing the difficulties of conventional blood sampling and practically applying it to a health examination.
Description
본 발명은 머리카락 절편 내 콜레스테롤 화합물들의 농도분석에 의한 계절별 변화 평가와 그의 임상적 응용 가능성에 관한 것이다.
The present invention relates to the evaluation of seasonal changes by analysis of concentration of cholesterol compounds in a hair slice and its clinical applicability.
분석 대상물질 중 콜레스테롤은 인체의 세포막을 구성하는 주요 구성요소로, 체내의 적절한 농도의 콜레스테롤은 대사과정에 필수적이지만, 지나친 양의 콜레스테롤은 혈관 내막에 침착하여 죽상동맥경화증의 원인이 되며, 원활한 혈액공급을 방해하여 심혈관질환을 유발한다. 또한 일반적으로 심혈관질환 고위험군 환자들의 경우, 혈액 내 콜레스테롤의 농도가 계절별로 큰 차이가 있다는 연구 결과가 보고된 바 있다(Altern Med Rev., 2005,10:307-320; Arch Intern Med., 2004,164:863-870). 하지만 계절별 콜레스테롤의 농도 변화를 통해 심혈관질환 고위험군을 예측하고자 하는 경우, 1년 동안 주기적으로 환자의 혈액을 채취해야 한다는 어려움이 있어 환자 친화적인 임상검진 방법에 속하지 못하는 단점이 있다. Cholesterol is a major constituent of the cell membrane of the human body. Cholesterol at an appropriate concentration in the body is essential for metabolism, but excessive amounts of cholesterol are deposited in the endothelium and cause atherosclerosis. It interferes with supply and causes cardiovascular disease. In addition, studies have shown that cholesterol concentrations in the blood are significantly different from season to high-risk patients with cardiovascular disease (Altern Med Rev., 2005, 10: 307-320; Arch Intern Med., 2004, 164: 863-870). However, when the high-risk group of cardiovascular disease is predicted through seasonal changes of cholesterol concentration, it is difficult to collect blood of patients periodically for one year, which is a disadvantage of not being a patient-friendly clinical screening method.
그러나 머리카락은 소변 및 혈액과 달리 평균 1개월에 1 ㎝씩 자라면서, 과거의 생리학적 정보를 포함하고 있기 때문에, 머리카락 1 ㎝안에 함유되어 있는 대사산물은 그 사람의 1개월간의 건강상태를 대변할 수 있을 것으로 보고되고 있다. 또한, 머리카락은 원하는 시기에 바로 시료 채취가 가능하고 보관방법이 용이하다는 장점이 있어 질환의 예측진단(J. Invest. Dermatol., 2001,116:57-61; Exp. Dermatol., 19:694-696) 및 인종간 생리학적 차이를 규명(J. Invest. Dermatol., in press)하기 위한 생체 시료로 유용하게 이용되고 있다. However, unlike urine and blood, hair grows 1 cm per month on average, and since it contains past physiological information, the metabolites contained within 1 ㎝ of hair represent the person 's 1 - month health status Is reported to be possible. Dermatol., 2001, 116: 57-61, Exp. Dermatol., 19: 694-7), since hair is readily sampled at a desired time and has a convenient storage method, 696) and to identify physiological differences between races (J. Invest. Dermatol., In press).
따라서, 혈액 채취의 단점을 해결하는 방법으로서, 머리카락을 이용하여 1년간 계절별 콜레스테롤 화합물들의 농도 변화 측정을 통해 관련 질환예측을 위한 환자 친화적인 검사방법이 요구된다.
Therefore, as a method for solving the disadvantages of blood sampling, there is a need for a patient-friendly examination method for prediction of related diseases through measurement of concentration change of cholesterol compounds for one year by using hair.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 인체 내 콜레스테롤 화합물 농도의 계절별 변화를 보다 안전하고 손쉬운 방법으로 측정하는 방법을 찾기 위해 노력하였다. 그 결과 종래의 주기적인 혈액 채취를 통하여 콜레스테롤 화합물 농도를 측정하는 방법과 달리, 머리카락을 이용할 경우, 지난 1년 간 성장한 한 올의 머리카락만으로도 각각의 계절별 콜레스테롤 화합물 농도의 변화율을 손쉽게 측정할 수 있으며, 이러한 변화율의 측정을 통해 관련 질환의 보유 여부, 발병 가능성 또는 질환의 호전 정도를 단 1회의 검사로 예측하는데 활용할 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventors have searched for a method for measuring the seasonal variation of the concentration of cholesterol compounds in the human body by a safer and easier method. As a result, unlike the conventional method of measuring the concentration of cholesterol compounds through periodic blood sampling, the rate of change of cholesterol concentration in each season can be easily measured by using only one hair of hair grown during the past year, The inventors of the present invention have completed the present invention by confirming that the measurement of the rate of change can be used for predicting the presence of the related disease, the likelihood of developing the disease, or the improvement of the disease with only one test.
따라서, 본 발명의 목적은 머리카락 내 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성에 대한 계절별 변화를 측정하는 방법을 제공하는 데 있다.Accordingly, it is an object of the present invention to provide a method for measuring seasonal changes in the concentration of cholesterol compounds in hair or metabolic enzyme activity of cholesterol compounds.
본 발명의 다른 목적은 상기 인체 내 콜레스테롤 화합물의 농도와 연관된 질환의 유무, 발병 가능성 또는 호전의 정도를 예측하는 방법을 제공하는 데 있다.
Another object of the present invention is to provide a method for predicting the presence or absence of disease, possibility of disease, or degree of improvement associated with the concentration of cholesterol compounds in the human body.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 다음의 단계를 포함하는 머리카락 내 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성에 대한 계절별 변화를 측정하는 방법을 제공한다:According to one aspect of the present invention, the present invention provides a method of measuring seasonal changes in the concentration of cholesterol compounds in the hair or the metabolic enzyme activity of a cholesterol compound, comprising the steps of:
(a) 머리카락을 성장한 계절별로 분리하는 단계;(a) separating the hair by season of growth;
(b) 각각의 계절별로 분리된 머리카락으로부터 콜레스테롤 화합물을 추출하는 단계; 및(b) extracting the cholesterol compound from each seasonally separated hair; And
(c) 상기 콜레스테롤 화합물의 계절별 변화를 측정하는 단계.
(c) measuring seasonal changes in the cholesterol compound.
본 발명자들은 인체 내 콜레스테롤 화합물 농도의 계절별 변화를 기존의 주기적인 혈액 검사보다 안전하고 손쉬운 방법으로 측정하는 방법을 찾기 위해 노력한 결과, 지난 1년 간 성장한 한 올의 머리카락에 계절별 콜레스테롤 화합물 농도의 변화에 대한 생리학적 정보가 그대로 담겨 있으며, 이를 콜레스테롤 관련 질환을 예측하는데 활용할 수 있음을 발견하였다.
As a result of efforts to find a method for measuring the cholesterol compound concentration in the human body in a safer and easier way than the conventional periodic blood test, the inventors have found that the change of cholesterol concentration It is found that the physiological information is contained as it is, and it can be used to predict cholesterol-related diseases.
이하, 본 발명의 측정방법을 각각의 단계별로 상세하게 설명하면 다음과 같다:Hereinafter, the measurement method of the present invention will be described in detail in each step as follows:
(a) 머리카락을 성장한 계절별로 분리 (a) Separation of hair growth by season
콜레스테롤 화합물의 계절별 변화를 측정하기 위해 먼저, 검사하고자 하는 대상으로부터 머리카락을 채취한다. 채취하는 길이는 계절별로 성장한 길이를 포함하고 있으면 무관하며, 바람직하게는 1년 이상 성장한 길이이고, 머리카락의 양은 측정하는데 필요한 양이면 무관하며, 바람직하게는 1년 이상 성장한 한 올의 머리카락이다. 머리카락은 성장기간은 사람에 따라 다르기는 하나, 평균 1 ㎝/월 성장하기 때문에, 봄, 여름, 가을 및 겨울 사계절의 정보를 습득하기 위해서는 약 12 ㎝이상의 머리카락을 이용하는 것이 좋다. To measure the seasonal changes of cholesterol compounds, hair is first taken from the subject to be examined. The harvesting length is irrelevant if it includes a seasonal growth length, preferably a length of more than one year, and the amount of hair is irrespective of the amount required for measurement, preferably an annual hair growth. Hair grows by 1 cm / month on average, although the growth period varies depending on the person, so it is better to use hair of about 12 ㎝ or more to acquire information of spring, summer, autumn and winter seasons.
이어서, 상기 머리카락을 각각의 계절별로 동일한 길이로 분리한다. 각각의 계절별 정보를 담고 있으면 그 길이는 무관하며, 바람직하게는 1 내지 3 ㎝가 적당하다. 상기 방법을 통해 각각의 계절별로 총 4개의 머리카락 절편을 수득한다.
Then, the hair is separated into equal lengths for each season. The length is irrelevant if it contains each seasonal information, preferably 1 to 3 cm. Through this method, a total of four hair segments are obtained for each season.
(b) 각각의 계절별로 분리된 머리카락으로부터 콜레스테롤 화합물 추출 (b) extracting cholesterol from each seasonally separated hair
이어서, 상기 4개의 절편을 이용하여 콜레스테롤 화합물을 추출한다. 추출에 적합하도록 머리카락을 분쇄하는 과정을 포함할 수 있으며, 티슈라이서(TissueLyser, Qiagen,USA)와 같은 장비를 이용하여 머리카락을 분쇄할 수 있다.The cholesterol compounds are then extracted using the four sections. The hair may be pulverized to be suitable for extraction, and the hair may be pulverized using a device such as a TissueLyser (Qiagen, USA).
콜레스테롤 화합물의 추출방법은 당업계에 알려진 다양한 방법을 활용할 수 있으며, 바람직하게는 고체상 추출방법을 이용하여 추출한다. 고체상 추출법을 이용할 경우 카트리지 안으로 추출물을 흘려주어 분석에 필요 없는 방해물질들을 제거할 수 있다. 추출용매로는 알코올을 이용하는 것이 바람직하며, 보다 바람직하게는 메탄올을 용매로 이용하여 콜레스테롤을 화합물을 용출한다.Various methods known in the art can be used for the extraction of cholesterol compounds, and the extraction is preferably performed using a solid phase extraction method. When solid-phase extraction is used, the extract can be poured into the cartridge to remove unwanted interferences. As the extraction solvent, alcohol is preferably used, and more preferably methanol is used as a solvent to elute the cholesterol compound.
상기 용출된 콜레스테롤을 화합물은 콜레스테롤, 콜레스테롤 전구체 및 콜레스테롤 대사체를 포함할 수 있으며, 보다 구체적으로는 데스모스테롤(desmosterol), 라노스테롤(lanosterol), 라토스테롤(lathosterol), 7-디하이드로콜레스테롤(7-dehydrocholesterol), 콜레스테릴 라우레이트(Cholesteryl laurate), 콜레스테릴 미리스테이트(Cholesteryl myristate), 콜레스테릴 팔미테이트(Cholesteryl palmitate), 7α-하이드록실 콜레스테롤(7α-Hydroxyl cholesterol), 프레그네놀론(pregnenolone)등을 포함할 수 있다.
The eluted cholesterol compound may include cholesterol, a cholesterol precursor, and a cholesterol metabolite, and more specifically, desmosterol, lanosterol, lathosterol, 7-dihydrocholesterol 7-dehydrocholesterol, cholesteryl laurate, cholesteryl myristate, cholesteryl palmitate, 7? -Hydroxyl cholesterol, (pregnenolone), and the like.
(c) 콜레스테롤 화합물의 계절별 변화 측정 (c) Seasonal change measurement of cholesterol compounds
상기 추출된 콜레스테롤 화합물들의 계절별 변화율을 측정하기 위해 각각 질량을 분석한다. 질량의 분석은 당업계에 공지된 다양한 질량분석기를 활용할 수 있으며, 바람직하게는 기체 크로마토그래피-질량분석기를 활용할 수 있다. 기체 크로마토그래피-질량분석기를 이용할 경우 시료의 휘발성을 증가시키기 위해 콜레스테롤 화합물들을 유도체화 시키는 과정이 추가될 수 있으며, 바람직하게는 MSTFA(N-methyl-N- trifluorotrimethylsilyl acetamide)/NH4I(ammonium iodide)/DTE(dithioerythritol) 혼합용액을 이용하여 유도체화 시킬 수 있다. 상기 방법을 통해 트리메틸실릴 유도체화된 시료는 휘발성이 증가하여 본 기체 크로마토그래피-질량분석기를 이용한 분석에 적합하게 된다.The mass of each of the extracted cholesterol compounds is analyzed in order to measure a seasonal change rate. The analysis of the mass may utilize various mass spectrometers known in the art, and preferably a gas chromatography-mass spectrometer can be utilized. When a gas chromatography-mass spectrometer is used, a process of derivatizing cholesterol compounds to increase the volatility of the sample may be added. Preferably, MSTFA ( N- methyl- N -trifluorotrimethylsilyl acetamide) / NH 4 I ) / DTE (dithioerythritol) mixed solution. Through this method, the trimethylsilyl derivatized sample is increased in volatility and is suitable for analysis using this gas chromatography-mass spectrometer.
또한, 본 발명의 측정방법은 콜레스테롤 화합물의 대사 효소 활성을 측정하는 단계를 추가적으로 포함할 수 있다. 상기 대사 효소는 7-환원효소, 24-환원효소 및 P450scc(cholesterol side-chain cleavage enzyme) 등이 있으며, 이러한 효소들의 활성 정도를 상기 계절별 콜레스테롤 화합물의 농도변화 정도와 비교하여 보다 상세한 계절별 변화율을 측정할 수 있다.
In addition, the measuring method of the present invention may further include a step of measuring the metabolic enzyme activity of the cholesterol compound. The metabolic enzymes include 7-reductase, 24-reductase, and P450scc (cholesterol side-chain cleavage enzyme). The degree of activity of these enzymes is compared with the degree of change in the concentration of cholesterol can do.
본 발명의 다른 양태는 상기 측정방법을 포함하는 인체 내 콜레스테롤 화합물의 농도와 연관된 질환의 유무, 발병 가능성 또는 호전의 정도를 예측하는 방법을 제공하는 데 있다.Another aspect of the present invention is to provide a method for predicting the presence or absence of a disease associated with the concentration of a cholesterol compound in the human body, including the above-described method of measurement, and the degree of the likelihood or the degree of improvement.
본 발명의 측정방법을 통하여 측정된 계절별 콜레스테롤 화합물의 농도 수치는 체내 콜레스테롤 수치 관련 다양한 임상적 응용 가능성을 갖는다. 예를 들어, 콜레스테롤 수치는 비만, 당뇨 등의 대사질환과 연관성이 높으며, 고혈압, 동맥경화증 등 다양한 심혈관 질환의 원인이 된다. 특히, 심혈관 질환 환자 또는 고위험군의 경우 혈액 내 콜레스테롤 화합물의 농도가 정상인과 비교하여 계절별로 큰 차이를 보이는데, 지난 1년간의 콜레스테롤 화합물 수치에 대한 생리학적 정보를 모두 포함하는 머리카락을 이용할 경우 1년 간 주기적으로 환자의 혈액을 채취해야 하는 혈액검사의 방법과 달리 한 올의 머리카락만으로도 계절별 콜레스테롤 화합물의 농도 변화를 매우 정확한 수치로 측정하여 이를 심혈관 질환의 보유 여부, 발병 가능성 또는 호전의 정도를 예측하는데 활용할 수 있다.The measured levels of cholesterol compounds by the measurement method of the present invention have various clinical application possibilities related to cholesterol levels in the body. For example, cholesterol levels are associated with metabolic diseases such as obesity and diabetes, and cause various cardiovascular diseases such as hypertension and arteriosclerosis. Particularly, in patients with cardiovascular disease or high-risk patients, the concentration of cholesterol in the blood is significantly different from that of normal people. When hair containing all of the physiological information on the level of cholesterol in the past one year is used, Unlike blood tests that require periodic blood sampling, Hanol's hair alone can be used to measure changes in serum cholesterol concentrations at very precise levels and to predict the presence of cardiovascular disease, the likelihood of disease, or the degree of improvement. .
예를 들어, 검사하고자 하는 대상의 머리카락에 존재하는 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성이 심혈관 질환을 보유하고 있지 않거나 발병가능성이 낮은 군으로부터 분리한 머리카락에 포함된 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성과 비교하여 계절별 변화율이 클 경우 심혈관 질환이 존재하는 군 또는 발병 가능한 군으로 판단할 수 있다.For example, the concentration of the cholesterol compound present in the hair of the subject to be examined or the concentration of the cholesterol contained in the hair isolated from the group in which the metabolic enzyme activity of the cholesterol compound does not have or does not have a cardiovascular disease, If the seasonal change rate is greater than the metabolic enzyme activity of the compound, it can be judged as a group in which cardiovascular disease is present or a group capable of causing the disease.
따라서, 본 발명을 활용하면 극미량의 머리카락으로부터 1년간 각 계절별 콜레스테롤류 화합물의 농도 변화 측정을 통해 심혈관질환 고위험군에서 나타나는 것과 같은 계절별 변화를 평가할 수 있으며, 특히, 콜레스레롤뿐만 아니라 관련 대사체들을 분석함으로서 대사관련 효소들의 활성을 함께 평가하여, 질환의 종류에 따라 다양한 예측 지표를 제공할 수 있다.
Therefore, by using the present invention, it is possible to evaluate seasonal changes such as those appearing in a high risk group of cardiovascular diseases by measuring changes in concentration of cholesterol compounds at each season for a year from trace amounts of hair, and particularly, By evaluating the activity of metabolic enzymes together, various predictive indicators can be provided depending on the type of disease.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(i) 본 발명은 머리카락 내 콜레스테롤 화합물의 농도 또는 콜레스테롤 화합물의 대사 효소 활성에 대한 계절별 변화를 측정하는 방법을 제공한다.(i) The present invention provides a method for measuring seasonal changes in the concentration of cholesterol compounds in the hair or metabolic enzyme activity of cholesterol compounds.
(ⅱ) 또한, 본 발명은 상기 인체 내 콜레스테롤 화합물의 농도와 연관된 질환의 유무, 발병 가능성 또는 호전의 정도를 예측하는 방법을 제공한다.(Ii) In addition, the present invention provides a method for predicting the presence, the possibility of disease or the degree of improvement associated with the concentration of cholesterol compounds in the human body.
(ⅲ) 본 발명에 따르면, 한 올의 머리카락만으로도 인체 내 콜레스테롤 화합물 농도의 계절별 변화를 매우 정확히 측정하여 기존의 주기적인 혈액 검사보다 안전하고 손쉬운 방법으로 측정할 수 있다.(Iii) According to the present invention, seasonal changes of the concentration of cholesterol compounds in the human body can be measured very precisely with a single-stranded hair, thereby making it possible to measure it more safely and easily than conventional periodic blood tests.
(ⅳ) 또한, 본 발명은 기존의 혈액 시료 채취의 어려움을 보완하고, 실질적으로 건강검진에 적용하여 보다 쉽게 콜레스테롤 대사 변화와 관련되는 질환들을 예측할 수 있는 장점을 갖는다.
(Iv) The present invention also has an advantage of being able to predict diseases associated with cholesterol metabolism change more easily by supplementing the difficulties of conventional blood sampling and practically applying it to a health examination.
도 1은 본 발명에서 머리카락을 활용하여 계절별 변화를 측정하기 위한 콜레스테롤 및 관련 대사체들의 화학적 구조를 나타낸다.
도 2는 본 발명을 통하여 머리카락 내에 존재하는 1년 동안 각 계절별 콜레스테롤 및 관련 대사효소들의 변화를 나타낸 결과이다.Figure 1 shows the chemical structure of cholesterol and related metabolites for measuring seasonal changes using hair in the present invention.
FIG. 2 is a graph showing changes in cholesterol and related metabolic enzymes during each year in hair according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example
실험 디자인Experimental design
본 발명은 한 올의 머리카락을 모근으로부터 3 ㎝씩 총 4개의 절편으로 나눈 후, 분쇄 및 추출 과정을 통해 콜레스테롤을 포함한 체내 대사산물들을 추출한 다음, 혼합성 고체상 추출 - 단백질 침전법을 이용하여 방해물질들을 제거함과 동시에 분석하고자 하는 콜레스테롤 화합물들을 정제한 후, 트리메틸실릴 유도체 반응을 거쳐 기체 크로마토그래피-질량분석기로 그 농도를 검출, 1년 동안의 계절별 농도 변화 양상을 평가한다.
The present invention relates to a method for separating cholesterol-containing metabolites from cholesterol by dividing a total hair into 3 fragments by 3 cm from the hair roots, And the cholesterol compounds to be analyzed are purified. The trimethylsilyl derivative is reacted, and its concentration is detected by a gas chromatography-mass spectrometer, and the seasonal variation pattern of concentration for one year is evaluated.
실시예 1Example 1
1) 분쇄 및 추출1) Grinding and extraction
채취된 머리카락은 모근으로부터 3 ㎝씩의 길이로 총 4개의 절편으로 나눈 후, 표면에 묻어있는 오염물질을 제거하기 위해 각각의 머리카락 절편들을 이소프로필알콜에 담가 30초 동안 흔들어 세척하였다. 이 과정을 두 번 반복한 후, 시험관에 세척된 머리카락 절편과 메탄올 0.5 mL, 그리고 내부 표준물질 20 μL와 분쇄용 구슬 3개를 넣고 티슈라이서(TissueLyser, Qiagen,USA)를 사용하여 20분 동안 1초에 25번 빈도수로 흔들어 머리카락을 분쇄하였으며, 그 안에 포함되어 있는 체내 대사물질들을 동시에 추출하였다.
The collected hair was divided into 4 sections with a length of 3 cm from the hair follicle, and each hair piece was soaked in isopropyl alcohol and shaken for 30 seconds to remove contaminants on the surface. After repeating this procedure twice, 0.5 mL of washed hair sections, methanol, and 20 μL of internal standard and 3 pieces of beads for grinding were placed in a test tube and incubated for 20 min using TissueLyser (Qiagen, USA) The hair was shaken with a frequency of 25 times per second, and the metabolites contained in the hair were simultaneously extracted.
2) 고체상 추출법2) Solid phase extraction
고체상 추출방법은 hybrid SPE-Phospholipid 카트리지(30 ㎎/1 mL, Supelco, USA)를 사용했다. 카트리지에 안으로 상기 1)의 추출물을 흘려주어 지질과 같은 분석 방해물질들을 제거하였고, 이어서 1.5 mL의 메탄올을 흘려주어 콜레스테롤을 포함한 관련 대사물질들을 용출시켜 깨끗한 시험관에 받았다. 그 후, 용출액에 포함되어 있는 용매를 40℃에서 질소 증발기를 이용하여 증발시킨 후, 다시 P2O5/KOH를 이용하여 진공건조기에서 30분 이상 충분히 건조시켰다.For the solid phase extraction, a hybrid SPE-Phospholipid cartridge (30 mg / 1 mL, Supelco, USA) was used. The extracts of 1) above were poured into the cartridge to remove analytical disturbances such as lipids, followed by 1.5 mL of methanol to elute the relevant metabolites, including cholesterol, in a clean test tube. Thereafter, the solvent contained in the eluate was evaporated at 40 ° C using a nitrogen evaporator, and then sufficiently dried for 30 minutes or more in a vacuum dryer using P 2 O 5 / KOH.
3) 기체 크로마토그래피-질량분석법3) Gas Chromatography-Mass Spectrometry
더욱 효과적인 기체 크로마토그래피-질량분석법을 위해 트리메틸실릴 유도체화를 통해 시료의 휘발성을 증가시켰다. 즉, MSTFA(N-methyl-N- trifluorotrimethylsilyl acetamide)/NH4I(ammonium iodide)/DTE(dithioerythritol) (500:4:5, v/w/w) 혼합용액 40 μL를 넣고 60℃에서 20분간 반응시켜 유도체화를 진행하였다. For more effective gas chromatography-mass spectrometry, the volatility of the sample was increased through the derivatization of trimethylsilyl. That is, 40 μL of a mixed solution of MSTFA ( N- methyl- N -trifluorotrimethylsilyl acetamide) / NH 4 I (ammonium iodide) / DTE (dithioerythritol) (500: 4: 5, v / w / w) And the derivatization was carried out.
분석에 사용한 기체 크로마토그래피-질량분석기는 Agilent사의 5975C 질량 선택검출기(Mass Selective Detector)가 연결된 6890 시리즈 기체 크로마토그래피이며, 특성 이온만을 선택하여 검출하는 선택적 이온 탐지법(selected ion monitoring mode, SIM)을 사용하였다. 또한, 분석 컬럼은 폴리다이메틸실록세인(poly dimetylsiloxane)이 코팅된 스테인레스 재질로 만들어진 Agilent사의 MXT-1을 사용하였으며, 이 컬럼의 길이는 15 m, 내경은 0.25 ㎜, 필름 두께는 0.25 ㎛이었다. 주입부의 온도는 260℃였고, 10:1의 분할 주입 방식을 적용하여 2 μL의 시료를 주입하여 분석하였다. 오븐의 온도 조건은 다음과 같이, 260℃에서 3분간 유지시키고, 2℃/분의 속도로 330℃까지 올려 8분간 유지시킨 후, 다시 30℃/분 속도로 380℃까지 올려 3분간 유지시켰다. 분석물질의 이온화 방식은 70 eV의 전자 충격 방법(electron impact ionization, EI)를 적용하였으며, 이온화가 일어나는 이온원의 온도는 230℃, 검출기의 온도는 150℃였다. 콜레스테롤 화합물들은 각각의 표준물질을 이용하여, 2개의 특성 이온의 피크 높이 비율과 컬럼 내 머무름 시간을 비교하여 확인하였다.
The gas chromatograph-mass spectrometer used in the analysis was a 6890 series gas chromatography coupled with Agilent's 5975C Mass Selective Detector, and the selected ion monitoring mode (SIM) Respectively. The analytical column used was MXT-1 manufactured by Agilent Co., Ltd., which was made of stainless steel coated with polydimethylsiloxane. The column had a length of 15 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm. The temperature of the injection part was 260 ° C and analyzed by injecting 2 μL of sample by applying a 10: 1 split injection method. The temperature condition of the oven was maintained at 260 캜 for 3 minutes, raised to 330 캜 at a rate of 2 캜 / minute, held for 8 minutes, raised to 380 캜 at a rate of 30 캜 / minute and maintained for 3 minutes. The ionization method of the analyte was electron impact ionization (EI) of 70 eV. The temperature of the ion source at which the ionization occurred was 230 ° C and the temperature of the detector was 150 ° C. Cholesterol compounds were identified by comparing the peak height ratio of two characteristic ions with the retention time in the column using each standard material.
시험예Test Example 1 One
상기 실시예 1의 분석방법을 통하여 3 ㎝의 길이로 절편된 머리카락들에 존재하는 콜레스테롤 화합물들의 농도를 측정하였으며, 그 결과를 표 1에 나타내었다. The concentration of cholesterol compounds present in the hair cut into 3 cm lengths was measured through the analysis method of Example 1, and the results are shown in Table 1.
계절별 농도를 나타내는 각각의 절편에서 콜레스테롤이 변화하는 경향을 확인하였으며(도 2), 측정된 화합물들 중, 3종의 콜레스테롤의 전구체인, 데스모스테롤, 7-디하이드로콜레스테롤, 라노스테롤, 그리고 산화콜레스테롤인 7α-하이드록시콜레스테롤이 콜레스테롤과 동일한 경향의 변화를 나타내고 있음을 확인하였다.
Dihydrololesterol, lanosterol, and oxidized fatty acids, which are the precursors of the three kinds of cholesterol, among the measured compounds (Fig. 2) It was confirmed that cholesterol, 7? -Hydroxycholesterol, exhibited the same tendency as cholesterol.
cholesterol7-Dehydro
cholesterol
laurateCholesteryl
laurate
myristateCholesteryl
myristate
palmitateCholesteryl
palmitate
cholesterol7α-Hydroxyl
cholesterol
(3 ㎝)First section
(3 cm)
(3 ㎝)Second section
(3 cm)
(3 ㎝)Third intercept
(3 cm)
(3 ㎝)Fourth intercept
(3 cm)
시험예Test Example 2 2
또한, 콜레스테롤 화합물들의 농도 평가에 의하여 관련 대사효소들의 활성을 측정할 수 있는 바, 7-환원효소, 24-환원효소 및 P450scc(cholesterol side-chain cleavage enzyme) 효소의 활성을 콜레스테롤의 변화와 비교 평가하였으며, 그 결과는 도 2와 같다.
In addition, the activity of the relevant metabolic enzymes can be measured by evaluating the concentration of cholesterol compounds, and the activity of 7-reducing enzyme, 24-reducing enzyme and P450scc (cholesterol side-chain cleavage enzyme) The results are shown in FIG.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (15)
(a) 한 올의 머리카락을 성장한 계절별로 분리하는 단계;
(b) 각각의 계절별로 분리된 머리카락으로부터 상기 화합물을 추출하는 단계; 및
(c) 상기 화합물의 계절별 변화를 측정하는 단계.
Desmosterol, lathosterol, 7-dehydrocholesterol, 7? -Hydroxyl cholesterol and pregnenolone in the hair comprising the steps of: : ≪ / RTI > a method for measuring seasonal changes in the concentration of a compound selected from the group consisting of:
(a) separating hair of one year by growing season;
(b) extracting said compound from each seasonally separated hair; And
(c) measuring the seasonal variation of the compound.
The method according to claim 1, wherein the hair is separated from the hair by four segments that are grown by one season.
5. The method of claim 4, wherein the sections are separated by 1 to 3 cm.
2. The method of claim 1, wherein step (b) comprises grinding hair.
The method of claim 1, wherein the extraction is performed using alcohol.
The method according to claim 1, wherein the measurement of the concentration is performed using a gas chromatograph-mass spectrometer after derivatizing the extract.
The method according to claim 8, wherein the derivatization is performed using a mixed solution of MSTFA ( N- methyl- N -trifluorotrimethylsilyl acetamide) / NH 4 I (ammonium iodide) / DTE (dithioerythritol).
The method of claim 1, wherein the method is selected from the group consisting of desmosterol, 7-dehydrocholesterol, 7? -Hydroxyl cholesterol, and pregnenolone. Lt; RTI ID = 0.0 > metabolic < / RTI >
The method according to claim 10, wherein the metabolic enzyme is selected from the group consisting of 24-reductase, 7-reductase, 7? -Hydroxyase and P450scc (cholesterol side-chain cleavage enzyme) ≪ / RTI >
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020120121162A KR101426529B1 (en) | 2012-10-30 | 2012-10-30 | Evaluation of Seasonal Variations of Cholesterols in Hair Fragments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020120121162A KR101426529B1 (en) | 2012-10-30 | 2012-10-30 | Evaluation of Seasonal Variations of Cholesterols in Hair Fragments |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20140054985A KR20140054985A (en) | 2014-05-09 |
| KR101426529B1 true KR101426529B1 (en) | 2014-08-05 |
Family
ID=50886738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020120121162A KR101426529B1 (en) | 2012-10-30 | 2012-10-30 | Evaluation of Seasonal Variations of Cholesterols in Hair Fragments |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101426529B1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120035471A (en) * | 2010-10-05 | 2012-04-16 | 한국과학기술연구원 | Evaluation of metabolic differences between hair samples by quantitative steroid signature |
-
2012
- 2012-10-30 KR KR1020120121162A patent/KR101426529B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120035471A (en) * | 2010-10-05 | 2012-04-16 | 한국과학기술연구원 | Evaluation of metabolic differences between hair samples by quantitative steroid signature |
Non-Patent Citations (4)
| Title |
|---|
| Biomedical Chromatography, 2006, 20(10), 999-1003.(2006.03.) * |
| Biomedical Chromatography, 2006, 20(10), 999-1003.(2006.03.)* |
| WebMD Health News, Cholesterol levels change with the seasons. (2004.04.) * |
| WebMD Health News, Cholesterol levels change with the seasons. (2004.04.)* |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20140054985A (en) | 2014-05-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Moldoveanu et al. | Gas chromatography/mass spectrometry versus liquid chromatography/fluorescence detection in the analysis of phenols in mainstream cigarette smoke | |
| Gaspar et al. | Organic metabolites in exhaled human breath—A multivariate approach for identification of biomarkers in lung disorders | |
| Schettgen et al. | Hemoglobin adducts of ethylene oxide, propylene oxide, acrylonitrile and acrylamide–biomarkers in occupational and environmental medicine | |
| Chun et al. | GC/MS combined with chemometrics methods for quality control of Schizonepeta tenuifolia Briq: Determination of essential oils | |
| Carrizo et al. | Direct screening of tobacco indicators in urine and saliva by Atmospheric Pressure Solid Analysis Probe coupled to quadrupole-time of flight mass spectrometry (ASAP-MS-Q-TOF-) | |
| CN109061017B (en) | Method for evaluating cigarette perfuming uniformity | |
| Nakanishi et al. | Quantitative mass barcode-like image of nicotine in single longitudinally sliced hair sections from long-term smokers by matrix-assisted laser desorption time-of-flight mass spectrometry imaging | |
| Sleiman et al. | Rapid and sensitive gas chromatography–ion-trap tandem mass spectrometry method for the determination of tobacco-specific N-nitrosamines in secondhand smoke | |
| Saha et al. | Rapid and sensitive method for simultaneous determination of six carcinogenic aromatic amines in mainstream cigarette smoke by liquid chromatography/electrospray ionization tandem mass spectrometry | |
| Johnson et al. | Accurate assignment of ethanol origin in postmortem urine: liquid chromatographic–mass spectrometric determination of serotonin metabolites | |
| Grova et al. | Gas chromatography–tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure | |
| WO2013103841A1 (en) | METHODS FOR QUANTITATIVE CHIRAL DETERMINATION OF THE d- AND l- ENANTIOMERS OF AMPHETAMINE AND METHAMPHETAMINE | |
| KR101505064B1 (en) | Discrimination of sitosterolemia in a dried blood spot | |
| Pellegrini et al. | Development and validation of a gas chromatography–mass spectrometry assay for opiates and cocaine in human teeth | |
| Kaur et al. | Headspace stir bar sorptive extraction–gas chromatography/mass spectrometry characterization of the diluted vapor phase of cigarette smoke delivered to an in vitro cell exposure chamber | |
| KR102211890B1 (en) | Nicotine metabolites extraction from hair and nail using QuEChERS sample preparation | |
| Janicka et al. | Sensitive determination of isoprostanes in exhaled breath condensate samples with use of liquid chromatography–tandem mass spectrometry | |
| KR101426529B1 (en) | Evaluation of Seasonal Variations of Cholesterols in Hair Fragments | |
| Jiang et al. | Simultaneous determination of multiple components in cigarettes by mechanochemical extraction and direct analysis in real time mass spectrometry in minutes | |
| Fan et al. | Simultaneous determination of nicotine and its nine metabolites in human urine by LC–MS–MS | |
| Guardiola et al. | Selective gas chromatographic determination of cholesterol in eggs | |
| Rodríguez-Fernández et al. | Gas chromatography double focusing sector-field ICP-MS as an innovative tool for bad breath researchPaper presented at the 2001 European Winter Conference on Plasma Spectrochemistry, Lillehammer, Norway, February 4–8, 2001. | |
| Xia et al. | Measurement of nicotine, cotinine and trans-3′-hydroxycotinine in meconium by liquid chromatography–tandem mass spectrometry | |
| CN109633014B (en) | Method for measuring contents of 21 terpenoids in tobacco leaves and application thereof | |
| Choi et al. | Measurement of testosterone and pregnenolone in nails using gas chromatography–mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| FPAY | Annual fee payment |
Payment date: 20170728 Year of fee payment: 4 |
|
| FPAY | Annual fee payment |
Payment date: 20180703 Year of fee payment: 5 |
|
| FPAY | Annual fee payment |
Payment date: 20190702 Year of fee payment: 6 |