쿠싱증후군 환자에서 당 대사 이상 정도에 따른 인슐린 감수성과 인슐린 저항성의 변화

정인경,김성훈,정재훈,민용기,이명식,이문규,유형준,안규정,노정현,김동준,김광원 대한내분비학회 2003 Endocrinology and metabolism Vol.18 No.4

연구배경 당질 코르티코이드는 당 대사에 매우 중요한 호르몬으로 내인성 당질 코르티코이드 과다상태인 쿠싱증후군에서는 말초조직에서 인슐린 저항이 증가하고 이를 보상하고자 인슐린 분비의 증가로 고인슐린혈증이 동반된다고 보고되고 있다. 하지만 생체 내에서와 달리 시험관내에서는 췌도세포에 당질 코르티코이드를 장시간 처리하면, 인슐린 분비 및 생합성이직접적으로 억제됨이 확인된 바 있어 쿠싱증후군 환자에서 당뇨병의 원인으로는 아마도 말초조직에서 증가된 인슐린 저항성 뿐 아니라 이를 충분히 보상하지 못하는 췌장에서의 인슐린 분비 저하가 같이 동반되어있지 않을까 하는 가설을 세우게 되었고, 아직까지 당질코르티코이드가 당대사 이상을 일으키는 기전에 대해 쿠싱증후군을 당대사 정도에 따라 인슐린 감수성과 분비능을 분석한 연구는 없었기에 이를 알아보고자 하였다. 방법: 삼성서울병원에서 쿠싱증후군으로 진단 받은 환자 15명을 대상으로 하였다. 이에 대한 대조군으로는 쿠싱증후군 환자와 같은 성별 그리고 체질량지수를 갖은 15명의 건강한 성인을 대상으로 비교 하였다 쿠싱증후군 환자를 대상으로 경구당부하 검사를 통해 당대사 정도를 정상군, 내당능장애군, 그리고 당뇨병군으로 나눈 후 정맥 당부하 검사를 시행하여 각군의 인슐린 저항성과 인슐린 분비능의 지표를 비교하고, 수술 후 쿠싱증후군이 완치된 상태에서 수술 전후의 당대사 지표의 변화를 조사하였다. 결과: 1) 쿠싱증후군 환자 중 정상인은 20%, 내당능 장애는 27%, 그리고 당뇨병은 53%였다. 체질량지수, 나이, 그리고 발병 기간은 세 군간에 의미 있는 차이가 없었으나, 24시간 소변검사의 코르티솔 농도는 당뇨병군에서 의미있게 높았다. 2) 정맥당부하 검사 결과, 인슐린 감수성 지표인 Sl는쿠싱증추린」서 1.58±0.10[×10^(-4)(min^(-1)(μU/mL)^(-1)]로 정상 대조군의 3.37±0.49[×10^(-4)(min^(-1)(μU/mL)^(-1)]에 비해 의미있게 낮았으나(P=0.024), 쿠싱증후군 환자 중 NGT, IGT, DM 군간에 서로 통계적인 차이는 없었다. 3) SG는 정상 대조군과 쿠싱증후군 환자간에는 의미있는 차이가 없었고, 쿠싱 증후군에 있어서 당대사가 악화될수록 감소하는 경향을 보였으나 의미있는 차이는 없었다. 4) 인슐린 분비능의 지표인 AIRg는 정상인에 비해 전체 쿠싱증후군 환자의 경우 증가하는 경향을 보였으나 의미있는 차이는 없었다. 하지만 쿠싱증후군 환자중에서 당대사 상태에 따라 NGT군은 1299 (1297∼1310)(mu/g/min ×10^(-2))로 정상 대조군(368.9±98.6[mu/g/min ×10^(-2)]) 보다도 의미있게 높았고, DM군{202.2 (91.1~371.4) [mu/g/min ×10^(-2)}은 NGT군에 비해 의미있게 낮았다(P=0.0031). 5) 15명중 현재 완치 상태에 있는 6명에 대해 수술전과 후로 비교하였다. 수술 전 당대사 상태가 1명은정상, 1명은 내당능 장애, 그리고 4명은 당뇨병이었으나 수술 후 시행한 경구 당부하 검사상 모두 정상 당대사 상태를 보였다. 6) 수술 후 완치된 환자 6명에 있어 인슐린 감수성지표인 Sl는 수술전에 중앙값이 1.22[×10^(-4)(min^(-1)(μU/mL)^(-1)]로 대조군에 비해 의미있게 감고』어 있었으나(p.0.05), 수술후 10.95 [×10^(-4)(min^(-1)(μU/mL)^(-1)]로 정상 수준으로 회복되었고(P=0.0022), 인슐린 분비능을 나타내는 AIRg [mu/g/min ×10^(-2)] 값도 정상수준으로 회복되었다. 특히 인슐린 분비능의 회복양상은 혈당농도에 따라 판이하게 나타나서, 정상과 내당능장애 상태에 있던 2명은 수술전에 1201 [mu/g/min ×10^(-2)]로 증가되어 있던 AIRg 값이 수술 후 정상 수준으로 감소하였고, 수술 전에 당뇨병 상태에 있던 4명의 경우 245.9 [mu/g/min ×10^(-2)]로 인슐린 분비능이 감고il어 있었는데 이들은 수술 후 모두 정상 수준으로 증가되었다 (P=0.0286). 결론: 쿠싱증후군 환자에서 당대사 이상은 80%로 높은 유병률을 보였다. 모든 쿠싱증후군환자에서 인슐린 감수성은 정상인에 비해 저하되어 있어 말초조직의 인슐린 저항이 선행됨을 시사하며, 인슐린 분비능은 당대사의 정도에 따라 다르게 나타났는데, 정상 당대사군에서는 인슐린의 저항성을 극복할 만큼 정상 대조군보다 더 많은 양의 인슐린 분비를 하다가 고코르티솔혈증이 심할수록 인슐린 분비능의 감소로 당뇨병으로 진행됨을 확인할 수 있었고, 이런 인슐린 저항성과 인슐린 분비장애는 수술 후 다시 회복되는 가역적인변화를 보였다. Background: Glucocorticoid plays an important role in the control of carbohydrate metabolism. Patients with Cushing's syndrome have been reported to have an increased incidence of carbohydrate intolerance due to peripheral insulin resistance and hyperinsulinemia, although the exact incidence and nature of this disorder have remained unclear. Few results have been published about insulin resistance and insulin secretion according to the level of glucose concentration, or about the reversibility of such defects in patients with Cushing's syndrome. Methods: To assess the effect of glucocorticoid on the insulin sensitivity and insulin secretion in Cushing's syndrome, 15 patients with Cushing's syndrome were classified into 3 groups (normal glucose tolerance: NGT, impaired glucose tolerance: IGT, diabetes: DM) according to the degree of glucose tolerance based on the oral glucose tolerance test (OGTT). Insulin modified, frequently sampled, intravenous glucose tolerance test (FSIGT) was performed before and after curative surgery on these patients and on 15 healthy control subjects. Data were evaluated by non-parametric statistical analysis. Results: 1) Among the 15 patients with Cushing's syndrome, 3 (20%) were NGT, 4 (27%) IGT, and 8 (53%) DM, based on OGTT. Twenty-four hour urinary free cortisol (UFC) was significantly higher in the DM group. 2) Insulin sensitivity index (SI) of Cushing's syndrome was significantly lower than that of the control group p=0.0024), but was not significantly different among the three Cushing's syndrome groups of NGT, IGT and DM. 3) Glucose mediated glucose disposal (SG) (Ed- confirm this abbreviation; it does not seem to match the definition) of Cushing's syndrome was not significantly different from that of the control group. 4) Insulin secretion (AIRg) of Cushing's syndrome tended to be high, but it was not significantly different from that of control. However, according to the level of glucose concentration there was significant difference in AlRg among the three Cushing's syndrome groups p=0.0031); AIRg of DM was significantly lower than that of NGT. 5) After surgical treatment, parameters of insulin sensitivity and insulin secretion were normalized in 6 cured patients; 1 with NGT, 1 with IGT, and 4 with DM, preoperatively. Median SI of all 6 patients was significantly improved up to the normal range postoperatively p=0.0022). Median AIRg of these 6 patients was balanced around that of normal control postoperatively p=0.0286). Conclusion: Eighty percent of patients with Cushing's syndrome had abnormality of carbohydrate metabolism. Insulin sensitivity was significantly decreased in Cushing's syndrome. Insulin secretion was significantly higher only in the NGT and IGT groups of Cushing's syndrome. As the hypercortisolemia is exacerbated, insulin secretion is significantly decreased and causes DM, suggesting that glucocorticoid has a direct or indirect toxic effect on the pancreatic beta cell (J Kor SOC Endocrinol 18:392-403, 2003).

신장기능영상용 방사성의약품 Bz - MAG3 ( Benzoylmercaptoacetylglycylglycylglycine ) 의 키트화 및 체내분포

고창순(Chang Soon Koh),이명철(Myung Chul Lee),정준기(June Key Chung),이동수(Dong Soo Lee),김영주(Young Ju Kim),정재민(Jae Min Jeong),장영수(Young Soo Chang),조정혁(Jung Hyuk Cho) 대한핵의학회 1996 핵의학 분자영상 Vol.30 No.3

N/A The MAG3 is a tubular excreting radiopharmaceutical for renal image. We synthe-sized benzoyl MAG3 (Bz-MAG3) and made a kit for labeling with Tc-99m. We checked the labeling effeciency of Tc-99m labeled MAG3 and biodistribution. Labeling efficiency was checked by TLC- SG (acetonitrile/H2O=2/1). After injecting of 1 mCi of Tc-99M-MAG3 to ICR-mice, Tmax(min), T1/2(min) were obtained in the renogram. Sequencial images (30sec, 2min, 5min, 10min, 15min, 20min) of TC-99m-MAG3 were compared with those of commercial Tc-99m-DTPA (Du Pont Merck Pharmaceutical Co.) kit. 1) The Rf value of synthesized Tc-99m-MAG3 was 0.78 and labeling efficiency was 97.5±1.9% (n=10). 2) The dynamic images of the Tc-99m-MAG3 were better than those of the Tc-99m-DTPA. 3) The Tmax(min.) and T1/2(min.) of Tc-99m-MAG3 (n=10) were 1.5±0.5 (left), 1.4±0.4(right), and 4.3±1.4 (left), 4.8±2.0 (right), respectivel. The Tmax(min.) and T1/2(min.) of Tc-99m-DTPA (n=7) were 2.7±1.6 (left), 2.7±1.6 right), and 3.8±1.7 (left), 4.5±2.7 (right), respectively. The quaility of image and labeling efficiency of the synthesized Bz-MAG3 kit were excellent, that it was supposed to be used in routine clinical work.

The Purification and Characterization of a Bacillus stearothermophilus Methionine Aminopeptidase (MetAP)

Chung, Jae-Min,Chung, Il-Yup,Lee, Young-Seek Korean Society for Biochemistry and Molecular Biol 2002 Journal of biochemistry and molecular biology Vol.35 No.2

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at $80^{\circ}C$, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at $80^{\circ}C$ for 45 min. The Km and Vmax values of the enzyme were 3.0mM and 1.7 mmol/min/mg, respectively. The B. stearothernmophilus MetAP was completely inactivated by EDTA and required $Co^{2+}$ ion(s) for activation, suggesting the metal dependence of this enzyme.

The Purification and Characterization of a Bacillus stearothermophilus Methionine Aminopeptidase ( MetAP )

Lee, Young Seek,Chung, Il Yup,Chung, Jae Min 생화학분자생물학회 1989 BMB Reports Vol.35 No.2

Methianine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at 80℃, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at 80℃ for 45 min. The Km and Vmax values of the enzyme were 3.0 mM and 1.7 mmol/min/㎎, respectively. The B. stearothermophilus MetAP was completely inactivated by EDTA and required Co^(2+) ion(s) for activation, suggesting the metal dependence of this enzyme.

원인 불명의 실신 환자의 진단에 있어서 Head - up tilt Test 의 유용성과 혈관미주신경성 실신 환자의 임상적 특징

윤호중(Ho Joong Youn),정욱성(Wook Sung Chung),백상홍(Sang Hong Baek),김철민(Chul Min Kim),박인수(In Soo Park),김재형(Jae Hyung Kim),최규보(Kyu Bo Choi),홍순조(Soon Jo Hong) 대한내과학회 1994 대한내과학회지 Vol.47 No.2

N/A Background: Syncope, defined as a transient loss of consciousness, is a frequently encountered symptom, but despite thorough clinical and invasive diagnostic investigation, the cause of syncope remains unexplained. In recent years, head-up tilt test has been of increasing interest as a diagnostic aids in patients with unexplained syncope. We studied to define the usefulness of various diagnostic test in the evaluation of patients with recurrent syncope, to assess the usefulness of head-up tilt test in the working of syncope of unknown origin and to evaluate the clinical characteristics of subgroups of patients with syncope induced by head-up tilt test. Methods: Between June, 1991 and November, 1992 at St. Mary`s hospital, 21 patients with history of syncope and 24 control subjects without history of syncope underwent 60° head-up tild test for 60 min. Results: 1) The definite cause for recurrent syncope were diagnosed in 10 of patients (47,6%) after clinical and invasive tests. 2) During head-up tilt test, vasovagal responses were provoked in 5 of 21 patients (23.8%) with recurrent syncope and 1 of 24 patients (4.2%) without syncope (p<0,05). 3) The head-up tilt test induced symptomatic bradycardia or hypotension in 3 of 10 patients (30%.) with negative electrophysiologic results. 4) During tilt induced vasovagal response, a) mean heart rate decreased to 42±24 beat/min from supine control of 70±12 beat/min (p<0. 05), b) mean systolic blood pressure decreased to 95±12 mmHg from supine control of 120±S mmHg (p<0,05), c) mean diastolic blood pressure decreased to 60±21mmHg from supine control of 72±13 mmHg (p<0.05), d) the time interval to onset of vasovagal response was 22±12 min. Conclusions: 1) In a large proportion of patients with recurrent syncope, the diagnosis remains unexplained despite a neurologic and cardiologic investigation including an electrophysiologic study. 2) Vasovagal responses are frequent cause in patients with recurrent unexplained syncope. 3) The head-up tilt test may be a simple, safe and highly yielding provocative test in the investigation of syncope of unknown origin.

A study of ^99mTc-sestamibi labeling condition using radio-chromatography

Moon, Sung-Hyun,Lee, Yun-Sang,Lee, Dong Soo,Chung, June-Key,Jeong, Jae Min Korean Society of Radiopharmaceuticals and Molecul 2017 Journal of radiopharmaceuticals and molecular prob Vol.3 No.1

Tc-99m labeled sestamibi ($^{99m}Tc$-MIBI) is one of most widely used radiopharmaceuticals for myocardial SPECT imaging. Radiolabeling of $^{99m}Tc$-MIBI is recommended by heating in $100^{\circ}C$ water bath for 15 min. However, the water bath might be a source of contamination. Thus, if radiolabeling of $^{99m}Tc$-sestamibi can be performed at room temperature, then it would be more convenient to use in clinical application. In this study, we performed the radiolabeling of $^{99m}Tc$-MIBI in different temperature conditions or using different instruments to find out the efficient labeling condition. We studied the $^{99m}Tc$-MIBI labeling at room temperature or $100^{\circ}C$ heating block, and checked the labelling yields every 1 min for 10 min using radio-TLC with 2 different eluents-saline and acetone. From the experiment, we confirmed that the $^{99m}Tc$-MIBI can be labeled over 90% yield but not completed at room temperature. However, the $^{99m}Tc$-MIBI labeling was completed when it was performed in the $100^{\circ}C$ heating block. Finally, we proved that heating is essential for complete $^{99m}Tc$-MIBI labelling, furthermore using heating block is also possible instead of water bath.

갑상선 기능항진증 환자에서 인슐린감수성 및 인슐린분비능의 변화

최은진,강호철,이태희,정민영,이대호,장연진,박상선,조재현 대한내분비학회 1994 Endocrinology and metabolism Vol.9 No.2

The impairement of glucose metabolism is frequently associated in hyperthyroidism. The present study was performed to determine the effect of the thyroid hormone excess on insulin sensitivity and on insulin secretory function in vivo. Ten newly diagnosed hyperthyroid patients and fifteen healthy control subjects were subjected to frequently sampled intravenous glucose tolerance tests(FSIGT) after an overnight fast. Insulin sensitivity, represented by the insulin sensitivity index(S_1), was assessed by minimal model analysis of FSIGT data. Insulin secretion was measured by the total area under the insulin curve after glucose load. The results were as follows. 1) The K_G values, which represent glucose tolerance, were not different between the hyperthyroid patients and the normals(2.2+-0.3 vs. 2.5+-0.3%/min, p$gt;0.05). 2) S_1 was significantly decreased in the hyperthyroid patients in comparison to the normals(7.5+-1.4 vs. 2.6+-0.3X10^-4 min^-1/uU/ml, p$lt;0.05). 3) The basal insulin concentration was higher in the hyperthyroid patients than in the normals(8.3+-2.4 vs. 4.6+-0.4 uU/ml, p=0.07). In addition, the insulin secretory response to a glucose load was increased in the hyperthyroid patients as evidenced by the peak plasma insulin level(168.2+-30.4 vs. 89.2+-13.9 uU/ml, p$lt;0.05) and by the total area under the insulin curve(2641.1+-443.2 vs. 1696.7+-204.3 min uU/ml, p$lt;0.05). These results clearly demonstrated that insulin sensitivity was impaired in these newly diagnosed hyperthyroid patients. However, glucose tolerance was maintained by the increased insulin secretion (J Kor Soc Endocrinol 9:108-114, 1994).

추출방법에 따른 감귤 과피 유래 Flavonoid의 추출효율 및 항산화 효과에 대한 비교

최찬익 ( Chan Ick Cheigh ),정원근 ( Won Guen Jung ),정은영 ( Eun Young Chung ),고민정 ( Min Jung Ko ),조상우 ( Sang Woo Cho ),이재환 ( Jae Hwan Lee ),장판식 ( Pahn Shick Chang ),박영서 ( Young Seo Park ),백현동 ( Hyun Dong Paik 한국산업식품공학회 2010 산업 식품공학 Vol.14 No.2

본 연구에서는 감귤 과피 가공부산물로부터 기능성 polyphenol 및 flavonoid가 새로운 추출방법인 아임계 추출법을 통해 추출되었고, 열수(80oC), 에탄올, 설탕용액을 이용한 기존 추출법과의 추출효율이 비교 분석되었다. 건조된 감귤 과피로부터 총 polyphenol(27.25±1.33mg QE/g DCP) 및 flavonoid(7.31±0.41mg QE/g DCP)에 대한 최대수율이 아임계 추출법(190°C, 1300 psi, 10 min)을 통해 획득되었으며, 이것은 기존 추출법 가운데 가장 높은 수율을 보인 에탄올 추출법을 통한 총 polyphenol(3.79±0.73mg QE/g DCP) 및 flavonoid(0.86±0.27mg QE/g DCP) 수율 대비 7.2배와 8.5배 이상 더 높은 것이었다. 추출방법에 따른 감귤 과피 추출물의 항산화 활성은 큰 차이를 나타내지 않았으나, 이러한 결과는 건조 감귤 과피 1g 당 아임계 추출법(190oC, 1300 psi, 10 min)에 의한 항산화 성분의 상대 수율(relative yield, %)이 다른 추출방법을 통해 획득된 것보다 대략 9.5배 이상 더 높다는 것을 설명하는 결과로, 아임계 추출법이 기존의 전통적 추출법에 비하여 감귤 과피의 기능성 polyphenol 및 flavonoid를 추출하는데 매우 적합한 방법임을 제시하고 있다. The extraction of polyphenol and flavonoid from citrus peel was performed by the ethanol, sugar, hot water (80oC), and subcritical water extraction methods. The maximum yields of total polyphenolic compounds (27.25±1.33mg QE/g DCP, QE and DCP indicate quercetin equivalent and dried citrus peel, respectively) and flavonoids (7.31±0.41mg QE/g DCP) were obtained by subcritical water extraction (SWE) with operating conditions of 190oC, 1300 psi, and 10 min. The yields by SWE were over 7.2-, and 8.5-fold higher than those of total polyphenols (3.79±0.73mg QE/g DCP) and flavonoids (0.86±0.27mg QE/g DCP) obtained using the ethanol extraction, which showed the highest extraction efficiency among tested conventional methods, respectively. Antioxidant activities of extracts obtained by different methods showed no significant differences. However, the relative antioxidant yield per 1 g dried citrus peel by SWE (190oC, 10 min) was over 9.5-fold higher than that by the ethanol extraction.

벤조디아제핀 수용체 영상용 양전자 방출 핵종 표지 플루마제닐 유도체 [F-18](3-(2-Fluoro)flumazenil의 합성과 생체 내 분포

장영수,이숙자,강삼식,홍성현,이명철,이동수,정준기,정재민,조정혁 대한핵의학회 1999 핵의학 분자영상 Vol.33 No.6

Purpose: Radiotracers that bind to the central benzodiazepine receptor are useful for the investigation of various neurological and psychiatric diseases. [C-11]Flumazenil, a benzodiazepine antagonist, is the most widely used radioligand for central benzodiazepine receptor imaging by PET. We synthesized 3-(2-[F- 18]fluoro)flumazenil, a new fluorine-18 (t1/2=110 min) labeled analogue of benzodiazepine receptor imaging agent, and evaluated in vivo for biodistribution in mice. Materials and Methods: Flumazenil (Ro 15-1788) was synthesized by a modification of the reported method. Precursor of 3-(2-[F-18]fluoro)flumazenil, the tosylated flumazenil derivative was prepared by the tosylation of the ethyl ester by ditosylethane. [F-18] labeling of tosyl substitued flumazenil precursor was performed by adding F-18 ion at 85℃ in the hot cell for 20 min. The reaction mixture was trapped by C18 cartridge, washed with 10% ethanol, and eluted by 40% ethanol. Bidistribution in mice was determined after intravenous injection Results: The total chemical yield of tosylated flumazenil derivative was ∼40%. The efficiency of labeling 3-(2-[F-18]fluoro)flumazenil was 66% with a total synthesis time of 50 min. Brain uptakes of 3-(2-[F-18]fluoro)flumazenil at 10, 30, 60 min after injection, were 2.5±0.37, 2.2±0.26, 2.1±0.11 and blood activities were 3.7±0.43, 3.3±0.07, 3.3±0.09%ID/g, respectively. Conclusion: We synthesized a tosylated flumazenil derivative which was successfully labeled with no-carrier-added F-18 by nucleophilic substitution.

개의 췌도분리에서 췌장의 팽창과 콜라겐 분해요소

이상훈,김성주,박정환,김종성,오승훈,한진수,정인경,양태영,김동준,김광원,이문규,민용기,이명식,정재훈,함종렬 대한당뇨병학회 2000 Diabetes and Metabolism Journal Vol.24 No.2

Backgrounds: One of the main problems conditioning the outcome of islet transplantation is the ability to separate a sufficient number of viable islets with preserved function. Islet purification is critically affected by all of the isolation stages. Thus, it is necessary to set up the standard isolation method that islets are separate well from acinar without compromising islet yield and viability. Methods: Twenty three adult mongrel dogs were used for the experiment of total pancreatectomy with islet isolation. The islets were properly isolated by a modified Recordi method. The obtained islets were further purified by centrifugation on discontinuous gradients using cell separation system (Model 2991, Cobe, Lakewood Colo). We evaluated islet number (islet equivalent number, 150 um equivalents/kg of recipient body weight, IEq/kg), purity, cell volume, viabilty, recovery rate, and comparison of outcome according to the isolation conditions. Results: 1) The mean of islet numbers before purification were 13543±9431Eq/kg, digestion times were 13.8±2.6 min., digestion temperature was 37.4±0.2℃, purity was 59.7?.0%, viability was 90.0±2.1%, cell volume was 4.7±1.1 mL, islet number after purification were 4064±361 IEq/kg, and recovery rate was 29±2.9%. 2) Isolated islet numbers were different according to the degree of pancreas distension with collagenase, digestion temperature, and digestion time. 3) The best conditions for islet isolation were above 37.5℃ in temperature at recirculation of collagenase, within 12 min in digestion time and well distended pancreas with collagenase. 4) According to multiple regression adjusted by variable factors, the degree of pancreas distension with collagenase and digestion time were independently associated factors for successful islet isolation. Conclusions: In this study, we concluded that the degree of pancreas distension with collagenase and digestion time were independent factors for successful islet isolation and the best conditions for islet isolation were above 37.5℃ in temperature at recirculation of collagenase, within 12 min in digestion time and well distended pancreas with collagenase.