Analysis of amino acid constituents of gallstones

Ying Chen, Lian Lian Wang,  Yu Xia Xiao,  Jing Hua Ni, Yan Yu

Subject headings  gallstones; amino acids/analysis; bilirubin; glycine

Chen Y, Wang LL, Xiao YX, Ni JH, Yu Y. Analysis of amino acid constituents of gallstones.
China Nati J New Gastroenterol, 1997;3(4):255-256

Abstract

AIM  To seek effective drugs to dissolve bilirubin, glycoprotein and black stones and better lithotriptics for cholesterol stones, and study the amino acid constituents of gallstones.

METHODS  According to the characteristics of infrared spectrum and the contents of bilirubin tested by semiquantitative chemical analysis, 30 of 148 cases of gallstones were selected and divided into 5 groups. Amino acids of the 30 cases were detected by high speed chromatography.

RESULTS  The quantity of amino acids in black stones was the highest (226.9mg/g) and that of pure cholesterol stones was the lowest, only 1.4mg/g in a case. In five groups of gallstones, the contents of amino acids were: black stone £¾ mixed bilirubin stone and glucoprotein stone £¾mixed cholesterol stone £¾pure cholesterol stone. The proportions were: 95.95¡Ã29.02 and 28.05¡Ã5.78¡Ã1. Aliphatic amino acids accounted for about 50% of the total amino acids in gallstones and  glycine 15.3% of the total amount of 17 kinds of amino acids.

CONCLUSION  In mixed stones, the higher level of bilirubin, the higher contents of amino acids. The acidic amino acids of bilirubin stones were relatively higher than those of cholesterol stones.



INTRODUCTION
Dissolution of cholesterol stones can be achieved by oral ursodeoxycholic acid or chenodehydrocholic acid, and by perfusion with methyl tertiary butyl ether as well. However, there has been no effective way to  dissolve bilirubin and glycoprotein and black stones. In order to seek better lithotriptics, contents of amino acids in gallstones have been studied.

MATERIAL AND METHODS

Sample selection
Gallstones in 148 cases obtained by surgery in our hospital during 1988-1992, were measured qualitatively with infra red spectrophotometer, 48 of them were also tested by semiquantitative chemical analysis. According to the characteristics of infrared spectrum and the constitents of bilirubin, 30 cases of gallstones were selected and divided into five groups: 10 pure cholesterol stones, 7 mixed cholestorol stones, 10 mixed bilirubin gallstones, 2 glycoprotein stones, and 1 black stone.

Sample treatment^£Û1£Ý
After pulverized by agate mortar and dried, 20mg powder of each gallstone was collected, added with 6mL of HCl (6mol), and sealed with nitrogen and baked at 110¡æ in oven for 24 hours. The volume was fixed to 25mL with distilled water. After filtration, 4mLwas dried in rotary evaporator and washed with distilled water twice. The remainings were dissolved in 2mL distilled water and 50¦Ìl or 100¦Ìl was used to measure the 17 amino acids, and taurine and ammonia. A trace of tryptophane was detected in two cases.

Analytical methods
Amino acids were detected by L-8500 high speed chromatography (Hitachi Corp, Japan). The column was 4.6mm¡Á60mm, using five buffer solutions for stepwise wash off, resin 2622 s.c. (Hitachi ion exchange resin was used). The standard amino acids samples were made by Sodium Glutamate Corp, Japan. The quantitative analysis was made with extensional calculation. The coefficient of variation (c.v.) was 1.5% in this experiment.

RESULTS
The various contents of amino acids of 30 gallstones in the five groups are illustrated in Table 1. There was glycine, glutamic acid, threonine, phenylalenine and ammonia in all the 30 cases, in which the glycine content was the highest, accounting for 15.34% of the total amount, glutamic acid came next, accounting for 13.01%. Asparagin, serine, valine, leucine, lysine,histidine and arginine were detected in 29 cases. There was a strong correlation (P£¼0.01) between the above-mentioned amino acids. In 29 cases, there was more acidic amino acid than alkaline amino acid (1.39-2.73¡Ã1), except one bilirubin mixed stone (1¡Ã1.77) which appeared like black mudy, and the patient had gallbladder malignant changes and liver metastasis after operation by pathology. The content of amino acids in one sample of pure cholesterol stones was the lowest in the 30 cases of gallstones (1.37mg/g). Six amino acids (glycine, glutamic acid, aspartic acid, serine, threonine, pyenylalanine), and taurine and ammonia were detected in this case. In 5 groups of gallstones, the constituents of amino acids of one case of black gallstone were the most complete, and the total amount of amino acids also was the highest (226.93mg/g), only the content of glycine was slightly lower than that of glycoprotein.
    In ten cases of pure cholesterol stones, the content of glycine was higher than that of glutamic acid and aspartic acid, and glutamic acid higher than aspartic acid, which were significantly different (P£¼0.005) statistically.
    In seven cases of mixed cholesterol stones, the content of glutamic acid was higher than that of aspartic acid, there was significant difference (P£¼0.01), but the content of glucine was higher than that of glutamic acid and aspartic acid, there was no significant difference (P£¾0.05).
   In ten cases of mixed bilirubin stones, although the content of glucine was higher than glutamic acid and aspatic acid, and glutamic acid higher than aspartic acid, there was no difference (P£¾0.05).
Table 1  The contents of various amino acids in 5 groups (30 cases) with gallstones    (x-¡Às, mg/g)

DISCUSSION
Amino acid is the fundamental unit of the protein constitution. Nowadays it is k nown that there are 20 kinds of amino acids^£Û2£Ý, which are controlled by genetic code in protein molecules. They are called living proteinic amino acids, which are glycine, alanine, valine, leucine, isoleucine, methionine, proline,  phenylalanine, tryptophan, tyrosine, serine, threonine, cysteine, glutamic acid , aspartic acid, histidine, lysine, arginine, asparagine and glutamine. In the 2 0 living protein amino acids,17 kinds of amino acids were detected, asparagine and glutamine were not, because during this experiment^£Û3£Ý, they would be  completely hydrolyzed to free aspartic acid and glutamic acid, leading to incre ased results. Moreover, the protein was heated and hydrolyzed with 6mol hydrochloric acid in the test, and tryptophan was damaged, and cystine, methionine, threonine and serine were also influenced, so the results were low. It was reported that cysteine only existed in bilirubin gallstones, but in our experiment, cysteine was not detected, and only a small amount of bisulfide compound of cysteine cystine and taurine produced during the conversion of cysteine in liver were detected.
    Our results indicate that protein amino acids generally existed in gallstones. The components of amino acids in gallstones are: aliphatic amino acid £¾ acidic amino acid £¾ alkaline amino acid £¾ aromatic amino acid £¾ suffur amino acid. In five groups of gallstones, the contents of amino acids were: black gallstone £¾ mixed bilirubin stones and glucoprotein stone £¾ mixed cholesterol stone £¾ pure cholesterol stone. Their proportions were: 95.95¡Ã29.02 and 28.05¡Ã5.78¡Ã1. The glycine was the most abundant in 8 kinds of aliphatic amino acids or 17 kinds of amino acids in gallstones. In mixed stones, the higher the content of bilirubin, the higher the content of amino acids. Except in the black gallstone, the amount of glycine was higher than that of aspartic acid and glutamic acid in all other gallstones. But glucine content was higher than aspartic acid and glutamic acid only in pure cholesterol stone(P£¼0.005), so it was clear that proportion of acidic amino acids in bilirubin stones was relatively higher than that of cholesterol gallstones. In 30 cases of gallstones, the components of acidic amino acids were higher than that of alkaline amino acids, except the bilirubin mixed stone in a patient with malignant changes of gallbladder and liver metastasis proved after operation by pathology. It is likely that the metabolic product of malignant tissue is not favorable for the stable existence of acidic amino acid. The mechanism awaits further studies.

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¹Department of Gastroenterology, Beijing Hospital, Beijing 100730, China
²Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China
This project won the third-class award by the Beijing Hospital for research achievements.
Correspondence to Dr. Ying Chen, female, born on 1942-12-14 in Shanghai, graduated from Nanjing Medical University in 1966, having 33 papers published.
Tel:+86¡¤10¡¤65132266-3135
Received  1996-10-26  Revised  1996-12-15