기질이 이당류 분해효소 적응성에 미치는 실험적 연구

김윤수,황석귀,송정석 ( Yoon Soo Kim,Suk Kuy Whang,Chung Suk Song ) 생화학분자생물학회 1975 BMB Reports Vol.8 No.2

In most mammals, levels of lactase are high during infancy but by the time adulthood is reached these levels of lactase are deficient. So far, the exact mechanism involved in the appearance and disappearance of lactase is not known. The present experimental study describes the digestive enzymatic adaptation of disaccharidase to the substrate in diet composition in order to investigate whether the mere presence of corresponding substrate in the diet suffices to stimulate disaccharidase or not, probably by $quot;turning on$quot; which encodes for the synthesis of these enzymes. Our experimental studies suggest that lactase activity in the newborn of the rat immediately after normal birth is at a peak and remains there through the lactating period; however, disappearance of lactase was observed four days after weaning. It reappeared in seven days after the rat, which had been fed for 150 days on a sucrose diet, was shifted to a lactose diet, However, the activity was weaker than that of the lactating period, indicating that enzymatic adaptation of latose to the substrate in diet may be related. On the other hand no surcease levels were found throughout the lactating period, but the levlel of surcease rose independently of the sucrose diet intake after weaning, indicating that the sucrose in the diet did not seem to influence the surcease activity at all.

Lactase Deficiency In Korean Adults

정명희,황석귀,김윤수,송정석,Chung, Myung-Hee,Whang, Suck-Kuy,Kim, Yoon-Soo,Song, Chung-Suk 생화학분자생물학회 1973 한국생화학회지 Vol.6 No.3

유당 분해 효소 lactase의 활성도는 일반적으로 분만과 동시에 최고치에 달하고 이유와 동시에 그 활성도는 서서히 떨어져서 성인에 이르르면 lactase 활성도는 민족에 따라서 다른 것으로 보고되고 있다. 그 원인이 그 민족의 환경 및 식사습성과의 관계 혹은 민족적 유전인자의 차이로 인한 것으로 설명하려 하고 있으나 어느쪽이 옳은 설명인지는 아직 모른다. 주위 환경과 음식 습성 및 민족의 기원이 상이한 한국인에 대한 이런 실험 보고는 아직 없기에 한국 성인을 대상으로 소정의 실험을 시도 하였다. 한국 성인에 유당을 부하하면 100%에서 유당분해효소 lactase가 결핍으로 오는 저혈당치를 보였으며 또한 한국성인에서 경구적으로 multipurpose rubin tube를 사용해서 얻은 생체 부검 조직내의 설탕분해 효소 sucrase는 정상치 인데 비하여 유당 분해 효소 lactase치는 실험대상 전원에서 그 활성도를 찾아 볼수 없었다. 이 원인이 한국인의 환경과 식이 습성에서 유래한 것인지 또는 조상때 부터 민족적인 어떤 유전인자 관계인지는 다른 민족에서 보고 된것 처럼 그 기전은 아직 모른다. Twenty six oral lactose tolerance tests were performed in 21 healthy subjects and five patients. Each subjects were given 50gm of lactose in 300ml of water. Venous blood sugar was measured at 0, 30, 60, 90 and 120 min. All 26 subjects showed a rise in blood sugar less than 25mg per 100ml (fiat curve) above the fasting level. At least one or more of G. I. symptoms such as bloating, abdominal cramps and/or diarrheas were experienced by all subjects during and/or after the tests. Additional glucose and galactose tolerance tests performed in 3 subjects were normal and this excluded the possibility of absorptive defect in these individuals. In order to determine the lactase activity of the Korean adults, peroral mucosal suction biopsies were done in another 10 subjects who were randomly selected. In all the subjects, the activities of lactase were absent whereas those of sucrase were normal. The majority, if not all, of Korean adults are expected to have lactase deficiency.

급성 및 만성 영양장애가 소화효소 활성 및 세포기능에 미치는 실험적 연구

김윤수,황석귀,김명원,유용운,송정석 ( Yoon Soo Kim,Suk Kuy Whang,Myung Won Kim,Yong Wun Ryu,Chung Suk Song ) 생화학분자생물학회 1974 BMB Reports Vol.7 No.1

A variety of digestive Enzymes of pancreatic and intestinal origin participate in the breakdown of protein, carbohydrate and lipids. It has been known that some of enzyme output of the intestinal glands can be manipulated by altering components of the diet. The incidence of patients who are suffering from gastrointestinal trouble in Korea is much higher than that of the United States. This fact might correlate with diet composition of Korean and American dishes. These facts are attractive for us to study the effect of diet composition on the digestive enzymes such as amylase, lipase, trypsin and chymotrypsin, and to decide whether the difference of diet composition affects these enzymes and whether such high incidence of gastroin testinal patients in Korea could be adequately explained by change in digestive enzyme activities of the animals fed with different diet. Our previous experiments have shown that pancreatic enzyme activites of rats fed with 5% casein diet which was prepared similarly to Korean diet in terms of protein content only were depressed as much as one fifth of that of control during two weeks feeding period. but it is true that protein is not only depleted but fat is also depleted in Korean dishes. The primary objective of the experiments described herein was to study the effect on the pancreatic enzyme activities of diet which was similarly prepared to Korean dishes and also to study the effect on the pancreatic enzyme activities of varying the proportion of fat to carbohydrate in the diet which is keeping the diet isonitrogenous and isocaloric. Data from this experiment will be discussed ire conjunction with previously published results conducted in our laboratory in order to illustrate how the high incidence of gastrointestinal patients in Korea could be adequately explained by diet prepared in similar composition to the Korean dishes. In a random distribution, male albino rats, weighing the average of 80 gm each were divided into five groups. Group I ; 5% casein : no fat : carbohydrate Group II ; 5% casein : 12% fat : carbohydrate Group III ; 18% casein : no fat : carbohydrate Group IV ; 18% casein : 12% fat : carbohydrate Group V ; 18% casein : high fat : carbohydrate (ratio of fat to carbohydrate 94 : 6) Calorie difference from protein or fat depletion was substitiuted by carbohydrate. At the end of designated time interval, the rats were sacrificed 14-15 hours after the last meal by decapitation. The pancreas was carefully romoved and placed in a cold 0. 25M sucrose solution (4℃). A 5% homogenate in 0.25M sucrose was prepared from the glandular {free of obvious lymph nodes and adipose tissue) portion of the pancreas. 5:% Homogenates were centrifuged (14,000 x g) for 15 minutes to remove cellular debris and whole cells. Enzyme analysis and protein determination were subsequently performed on the supernatant. Protein was determined by Lawry method (1951). Enzyme activity assays were performed using modification of Hepler method (1962) for amylase and the method developed by Cherry and Crandall (1932) for lipase. Chymotryptic assays were performed using a modification of method developed by Rhodes et al. (1957), as a substrate for chymotrypsinogen N-acetyl-L-tyrosine ethyl ester was used. Trypsinogen assay was similarly performed using p-toluene-sulfonyl-L-arginine methyl ester as a substrate. The assays were linear with respect to enzyme concentration over the range used under specified conditions such as temperature (37℃) and incubation period, 30 minutes for amylase, 1 hour for lipase, and for the trypsinogen and chymotrypsinogen the recording of dye disappearing of optical density was recorded 2 to 3 seconds after adding substrate-buffer-indicator The results of the experiments in which the effect of different composition of nutrient which was prepared in similar composition to Korean dishes on pancreatic enzyme acitivities are summarized as the following. When the enzyme activity

급성 및 만성 영양장애가 소화효소 활성 및 세포기능에 미치는 실험적 연구

김윤수,황석귀,김명원,유용운,송정석,Kim, Yoon-Soo,Whang, Suk-Kuy,Kim, Myung-Won,Ryu, Yong-Wun,Song, Chung-Suk 생화학분자생물학회 1974 한국생화학회지 Vol.7 No.1

저자들은 한국인 주식에 흡사한 각종 식이 조성을 달리한 인공 식이가 취장소화효소 활성에 어떻게 영향을 미치며 이들 식이 조성차이와 소화효소 활성과의 함수관계를 알아 보기 위하여 식이 조성을 달리한 각종 식이를 가지고 실험한바 다음과 같은 실험결과를 얻었다. 식이 I군(저단백, 무지방, 고함수탄소)과 식이 II군(저단백, 정상지방, 고함수탄소)으로, 4주 또는 6주동안 사육후 백쥐의 취장 소화 효소 활성을 측정하여 그 변화를 비교 관찰하였다. 정상식이(정상단백, 정상지방, 정상 함수탄소)로 해당 기간 동안 기른 쥐의 각 효소 활성치를 100%로 표시할 때 식이 I, 식이 II로 2주 동안 기른 쥐 취장 소화 효소 chymotrypsinogen은 정상치의 0~10% trypsinogen은 0~20% amylase는 10~15%, lipase 20~25%로 저하 하였다. 이와 같은 효소 활성치의 저하는 계속 4주 6주에도 유지 하였으며 식이 III군(정상단백, 무지방, 고함수탄소)에서는 정상군과 하등의 변화가 없이 소화효소 활성치를 유지하였다. 즉 소화효소 chymotrypsinogen, trypsinogen, amylase, lipase 이들 모든 효소 활성은 우리가 사용한 지방의 함량에 하등의 영향을 받지 아니 하였으며 식이중 단백질 함양유무(18%)가 효소활성 저하의 중요한 요인의 하나라는 사실을 실험적으로 입증하였다. 이와 같이 식이 조성 차이로서 저하된 각식이군의 효소 활성도는 4주, 6주에 정상식이로 대체 하였을 때 식이 I, II로 4주, 6주동안 사육한 기간에 상관없이 식이 대체 3일만에 chymotrypsinogen, trypsinogen, Iipase는 정상치로 회복하였으며 amylase는 4주에 대체한 것이 5일, 6주에 대체한 것이 7일을 요하였다. 즉 식이에 의한 amylase 효소 활성 회복 반응은 chymotrypsinogen, trypsinogen, lipase와 상이 하였다. 4, 6주후에 정상식이 대신 저단백(5%) 고지방(78%) 저함수탄소(17%)로 조성된 고 지방식이로 대체 하였을때 소화효소는 전혀 회복을 보이지 아니한 사실로 미루어 보아 소화효소 활성 회복에는 단백질함양이 절대필수요소 임을 실험적으로 입증하였으며 지방은 전혀 관계하지 않은 사실도 압증하였다. 정상단백질(18%)이 일정하고 고지방(78%) 저함수탄소(5%) 식이로 3주, 4주 동안 기른 쥐의 취장내 효소활성 chymotrypsinogen, trypsinogen, lipase는 하등의 영향을 받지 아니 하였으며 amylase 치만이 정상치 100% 보다 3주에 61.5%, 4주에 64%로 저하된 효소 활성치를 보였다. Amylase는 지방 함양보다 함수탄소양에 영향 받은 것 같다. 이상과 같은 우리의 실험적 사실은 취장 소화 효소 활성은 식이조성 차이로서 영향 받으나 지방 함양의 다소 유무에 하등의 영향을 받지 아니하고 오로지 단백질 함양 만이 그들효소의 활성 및 회복에 필수요소 임을 실험적으로 입중하였으며 amylase는 단백질 함양에도 영향 받으나 단백질 함양이 정상이고 함수탄소양을 줄인 고지방식이(함수탄소 : 지방 6 : 94)는 amylase 활성에 영향을 미친다는 사실도 구명하였다. the corresponding tissue fed with normal diet, the enzyme activities of chymotrypsinogen, trypsinogen, amylase and lipase were depressed by 0-10%, 0-20%, 10-15% and 20-25% respectively at the end of two weeks during the feeding of group I and group II and then these range of depletion of enzyme activities maintained continuously up to the end of tour and six weeks. This result suggests that digestive enzyme activities are influenced by only depletion of protein but not by various amount of fat in diet at all when carbohydrate is sufficient. Some of animals in groups I and II receiving designated diet for four and six weeks were transferred to the normal diet then pancreatic enzyme were assayed on the third, fifth and seventh days. The recovery of these enzymes is remarkable, and apparently the rats fed protein-and lipid-depleted diet were able to respond immediately to the stimulus of diet. Complete recovery of chymotrypsinogen, trypsinogen and lipase was observed after three days feeding regardless rats fed with protein- and lipid-depleted diet for four or six weeks except amylase which was recovered after five days feeding in the group of rats fed diet I and II for four weeks and seven days feeding in the group of rats fed diet I and II for six weeks. No difference in the recovery rate of enzyme was observed between diets I and II and also between the group of the rats when the rats in groups I and II shifted to the normal diet at four weeks and six weeks. Rats fed protein- and lipid-depleted for four and six weeks were not able to respond to the stimulus of diet, at all, which consists of high lipid. low protein, and low carbohydrate. This result illustrates that the essential factor for stimulus of enzyme activity is protein in diet but not carbohydrate and fat. The effect of high proportion of fat to carbohydrate (94: 6) in isonitrogenous, isocaloric diet on pancreatic enznyme activities also was studied in rats and compared to the effect of normal isocaloric diet. The diet which provided 57 cal/day and the experiment lasted for four weeks, no effect of this diet on pancreatic chymotrypsinogen, trypsinogen and lipase was observed. However, pancreatic amylase was reduced to 61.5% at the end of three weeks and 64% at the end of four weeks. This result indicates that pancreatic amylase activity may be related when carbohydrate is restricted but protein is normal in diet. These results indicate that when various dietary composition is related to the pancreatic enzyme activity, one of main influential factor in diet on pancreatic enzyme is protein, but not fat at all. However pancreatic amylase acivity may be related when carbohydrate is resticted but protein is normal in diet. Our experimental evidence tends to indicate that enzyme synthesis in the pancreas is regulated, at least in part by the composition of amino acid pool(s) in the acinar cells of pancreas. This may be due to the decreased amount of amino acid in the dietary protein, but not composition of fat. It can be postulated that the decrease in the dietary protein affects synthesis and secretion of pancreatic digestive enzymes, and then the intracellular structure of the cell, resulting in decrease of cell activity and tissue debility.

Dietary Induced Depletion and Repletion of Pancreatic Enzymes : Amylase and Lipase

김윤수,송정석,유용운,황석귀 생화학분자생물학회 1971 BMB Reports Vol.2 No.2

A variety of digestive enzymes of pancreatic and intestinal origin participate in the break down of protein, carbohydrate and lipids. It has been known that some of enzyme output of the intestinal glands can be manipulated by altering components of the diet. The incidence of patients who suffering from gastrointestinal trouble in Korea is much higher than that of United States. This fact might correlate with diet composition between Korean dishes and American dishes. These facts attractive for us to study the effect of diet composition on the digestive enzymes such as amylase, lipase, trypsin and chymotryosin, whether the difference of diet composition is effect on these enzymes and whether such high incidence of gastrointestinal patient in Korea. could be adequately explained by change in digestive enzyme activities of the animals fed with different diet. In a random distribution, male albino rats, weighing the average of 85 gm each were divided into three groups. The groups were designated as group I (5% casein diet), group II (18% casein diet), group III (25% casein diet). The rats were trained to eat each designated diet during 1 hr period every 12 hrs. For two weeks (28 feedings) the food intake of all rats was ad libitum. At the end of the 28 th feeding, and 2.5 hours after the beginning of the 1 hr feeding period, rats were Stilled by decapitation. The pancreas was carefully removed and placed in a cold 0.25 M sucrose solution (4 ) . A 10% homogenate in 0.25M .sucrose was prepared froth the glandular (free of obvious lymph nodes and adipose tissue) portion of the pancrens. The 10% homogenate was assayed for protein and enzyme activity. Protein was determined by micro -=Kjeldah1 method. Amylase activity assays were performed using a modification of Somogi's method. Lipase activity assays were performed using a modification of the method developed by Cherry and Crandall. We found that the standard curve of amylase and lipase were proportional to the enzyme concentration under specified conditions such as temperature, (37°) and incubation period, 30 minutes for amylase, 1 hr for lipase. Results of the experiments in which the effect of protein content on pancreas tissue enzyme activity are summarized as following. When the enzyme activity in the pancreas, expressed as %, in the corresponding tissue fed with 18% casein diet, enzyme activity of amylase and lipase were depressed by 12.5% and to 29.6% during the 28 feeding of the 5% casein diet, Some of animal in group I receiving 28 feedings were transferred to the 18% and 25% casein diet, then pancreatic tissue enzymes were assayed 2. 5 hr after the initiation of the 1 hr feeding period, on the 2nd day (4th feeding) 5th day (10th feeding) and 7th day (14th feeding) , The recovery of amylase and lipase activity were remarkable. This recovery was not seen until fourth feeding. It was remarkably rapid a#ter the fourth feeding to tenth feeding' From the tenth to the fourteenth feeding the enzyme activity slowly recovered to 80% activity for the 18% casein diet, No significant difference in the recovery rate of enzyme was observed between 18% casein diet, 25% casein diet, and 5% casein diet in the rats shifted to group II and group III diets. On the other hand, immediately after the first feeding there was recovery of Iipase enzyme activity, to about normal by the tenth feeding of the 25% caesin diet. However, the recovery of pancreatic lipase enzyme activity of rats 18% casein digit was not as rapid as on the 25% casein diet. These results suggest that there are remarkabls effects of diet on digestive amylase and lipase enzyme activities. This may be due to the decreased amout of amino acid in the dietary protein. It can be postulated that the decrease in dietary protein affects synthesis and secretion of pancreatic amylase and lipase, and then with the intracellular structure of the cell, resulting tissue debility. The effect of diet on trypsin and chymotrypsin activity of pancreatic tissue is still not understood. We are investigating how these enzymes are affected by diet. From our investigations, we have founded a remarkable relationship between the dietary protein content and pancreatic digestive enzyme activity. The depression of digestive enzyme activity of pancreas was not found after one or two high protein meals. It took at least ten or more meals to develop the reported effect.

Radio Immuno Assay of Insulin affected by Gelatin in Vivo and Vitro System

김윤수,송정석,전의철 생화학분자생물학회 1971 BMB Reports Vol.2 No.2

At the present time gelatin is widely in the dietary control of obesity because of its low caloric content. Recent studies in the pancreas of the rat have demonstrated that gelatin has a biosynthetic effect on trypsin and chymotrypsin. The mechanism of this effect is unknown. Studies in rabbits in our laboratory have demonstrated that gelatin also has an hyperinsulinemir. effect on glucose tolerance, and have led us to investigate possible answers to the following questions. (1) What is the component of gelatin which causes the development of the hyperinsulinemic glucose tolerance ? (2) Are there any quantitative relatioships between the glucose and insulin levels in the blood of rabbits receiving gelatin as compared with the controls ? (3) What is the effect on human beings of the hyperirsulinemic factor found in gelatin ? We postulated that gelatin then may contain a regulatory secretion factor which affects biosynthesis and pancreatic secretion of insulin. To search for this hypersecretory factor in gelatin a quantitative radioimmuno assay of insulin as it was affected by the factors of glucose and ZnCl₂ was made in vitro. Four experiments were done using 2 ㎏ rabbits. which had been fed bean curd refuse with fresh vegetables. In experiments I, after the animals were fasted for 12 hours, a glucose tolerance test (Exton-Rose method) was done on each. In Experiment II, the glucose tolerance test was done 2. 5 hours after the administration of 15 gm of gelatin in 30m1 of water by stomach tube. The control animals were given 30 ㎖ of water. In Experiment III, instead of the gelatin, the animals were given 200 ㎎ of zinc as ZnCl₂ in the 30 ㎖ of water. Blood for the measurement Of glucose was removed at 0, 30, 60, 90, 120 minutes intervals from the rabbit's ear vein. Plasma glucose was determined by the Folin-Wu micro method. Using the method proposed by C. N. Hales and P. J. Randle, a quantitative radioimmunoassay of the plasma insulin was made. ^(-125)I labelled, human insulin and ^(-125)I labelled pig insulin, Tong with insulin binding reagents, were obtained from the CEA-CEN-SORIN comany in France. A standard calibration curve was prepared. In Experiment IV, immediately after decapitation of the rabbit the pancreas was carefully removed and placed in a cold bicarbonate buffer solution (4 ). Obvious lymph nodes were removed from the glandular portion along with adipose tissue. The pancreas then was cut into R-11 ㎎ pieces. 500 ㎎ of pancreas, divides into small pieces, were blotted on filter paper and then placed in 2 ㎖ of the incubation medium. It is evident that, as the glucose level in the blood increased, the secretion of insulin also increased until a plateau was reached 60 minutes after the administration of glucose. From this 60 minutes plateau the insulin level returned to its original value in 60 minutes. However, the glucose level continued to remain high, as much as 170 ㎎ glucose. When glucose and gelatin were given to the rat at the same time there was no difference in the glucbse levels observed in the gelatin treated rats and the control rats. In remarkable contrast, however, rabbits given 15 gm of gelatin 2.5 hours before being given glucose showed a 30 minutes secretion level of insulin which was twice that of the control :animals. This occured even though the glucose level in the two groups remained the same over the 'same interval of time. Thus the effect is due directly to the gelatin. This fact was also true when 200 ㎍ of. ZnCl₂ was given instead of gelatin. Meanwhile no remarkable difference were observed in the glucose levels between the group given ZnCl₂ with glucose at the same time as gelatin and the controls. The maximal excretion sate came at a concentration of 2b ㎍/100㎖. If the ZnCl₂ concentration was increased to more than 26 ㎍/100㎖ concentration, in vitro, there was no corresponding increase in insulin secretion. This would correlate with in vivo experiments in which it was found that the insulin was not secreted at all when 40㎍ of ZnCl₂ was administering by orally to the 2 ㎏ of rabbit, these amount of ZnCl₂ was administered is .equivalent to 33 ㎍ of ZnCl₂ concentration/100 ㎖ of infra and extracellular fluid of rabbit. The study reported here was designated to provide us with information concerning the effect of gelatin and ZnCl₂ on insulin secretion in vivo and in vitro. We had a postulate as to which components of gelatin were involved in the secretion of insulin. The study partially confirmed the postulate that glucose, insulin and Zn levels in blood are related in vivo and in vitro. We are now investigating the relationships. between the glucose, insulin and Zn levels in vitro, at the present time this study is not complete.

Dietary Induced depletion and Repletion of Pancreatic Enzymes in the Rat with Emphasis on Korean Dishes

김윤수,김명원,송정석,황석귀 생화학분자생물학회 1978 BMB Reports Vol.6 No.3

The primary objectives of the experiments described herein were to study the effect on the pancreatic enzyme activities -of a diet which was similarly prepared to Korean dishes and also to study the effect on the pancreatic enzyme activities of varying the proportion of fats to carbohydrates in the diet which keeps the diet isonitrogenous and isocalories. The data from the this experiment will be discussed in conjunction with previously published results conducted in our laboratory. The incidence of gastrointestinal patients in Korea could be adequately explained by diets prepared in similar composition to the Korean dishes. Rats fed with protein and lipid-depleted diet for four and six weeks were unable to respond to the stimulus of the diet at all which consists of high lipid, low protein and low carbohydrate. This result illustrates that the essential factor for stimulus of enzyme activity is protein in diet but not carbohydrate and fat. The effect of high proportion of fat to carbohydrate (94 : 6) in a isonitrogenous, isocaloric diet on pancreatic enzyme activities also was studied in rats and compared with the effect of normal isocaloric diet. When a diet which provided 57 cal/day was fed for four weeks, no effect of this diet on pancreatic chymotrypsinogen, trypsinogen and lipase was observed. However, pancreatic amylase was 61.5% after three weeks and 64% after four weeks, indicating that pancreatic amylase activity may be related when carbohydrate was limited but protein was normal in diet. It is therefore suggested that various dietary composition was related with the pancreatic enzyme activities and that one of main influential factors in diet on pancreatic enzyme is protein, but not fat.