Biliary proteins have been suggested to play an important role in bile nucleation and gallstone formation. Supersaturation of gallbladder bile is the first step in gallstone formation¹⁾ and nucleation from supersaturated bile of calcium salts of cholesterol or bilirubinate have been implicated in gallstone formation²⁾. Normal bile contains factors that inhibit nucleation, whereas lithogenic biles contain factors that promate nucleation³⁾. Although the precise role of nucleation has not been well characterized, recent study suggest that these factors are probably proteins^4–6). It should be also suggested that nucleation requires an organic matrix, composed of small, soluble acidic regulatory proteins bound to a structural framework of high molecular weight gallbladder mucins.

Several investigators have reported results of measurements of total protein concentrations in biles of gallstone patients, but they differ in their opinions with different results^7–10). The idea that there might be a specific differences in the protein compositions in patients with cholesterol and pigment gallstones may provide information as to the nature of nucleating or antinucleating factors in bile. Little data are available, however, to compare the biliary protein patterns of patients with cholesterol or pigment stones and those without gallstones. We have tried to extract, isolate, and characterize the proteins in patients with gallstones and without gallstones.

Gallstones have become a major public health problem in all parts of the world¹⁶⁾, and the incidence has been increasing in the Orient, even in our country^17,18). There are two major classes of gallstones: cholesterol gallstones and pigment gallstones on the basis of their major composition, characteristics and pathogenesis¹⁹⁾. Cholesterol stones consist of at least 70% cholesterol, often with alternating layers of brown to black pigments, in which case they are called mixed stones²⁰⁾. Pigment stones are subclassified into two groups: black pigment stones and brown pigment stones²¹⁾. Black pigment stones contain much insoluble black pigment that is highly crosslinked network polymer or tetrapyrroles²²⁾, which is mixed with calcium salts of bilirubinate, carbonate and/or phosphate^11,21). Brown pigment stones consist mainly of the insoluble calcium bilirubinates, calcium soaps of fatty acids, and a significant proportion of cholesterol ^23,24).

The classification of gallstones has been based on morphological and chemical analysis but has been limited by the variety and insolubility of many of the components²⁴⁾. The introduction of solid state analytical techniques such as X-ray diffraction and infrared spectroscopy has permitted identification of insoluble components of gallstones^25,26,27). The infrared spectroscopy has an advantage that it could identify the components in pigments stones without requiring gallstones crystallinity and solubilization^22,23,28).

Gallstone pathogenesis can be broken down into three distinct stages: 1) supersaturation, 2) nucleation, and 3) stone growth. Early investigators focussed primarily on cholesterol saturation, and most studies revealed that normal bile may be supersaturated and pathological bile is always supersaturated. However, supersaturation is not a sufficient condition for nucleation and gallstone formation^7,29,30). Cholesterol monohydrate crystals precipitate much more rapidly from lithogenic biles than from normal biles, due to potent factors that promote nucleation of cholesterol³¹⁾. Normal bile also contains potent proteins that inhibit nucleation of cholesterol and calcium salts from supersaturated solutions including abnormal biles^3,5,32). These inhibitory proteins are diminished in the gallbladder bile of patients with gallstones^33,34). A great deal of interest has recently focused on the identification of the potential pronucleating factors.

In the present experiment, there was no statistical difference in protein composition among three types of gallstones. Harvey et al also reported that the glycoprotein concentrations in three types of gallstones were not significantly different³⁵⁾. The mean protein concentrations in our study were a little bit higher than those from Harvey's study and those determined by Lowry and Bradford³⁶⁾. However, the mean protein concentration was higher (47.6±25.9 mg/ml) in cholesterol gallstones than in pigment stones or no stones, and it is still possible that the onset of nucleation may be associated with a temporary increase in protein concentration which then returns to normal levels once nucleation has occurred.

As in most biomineralization processes in mammals, there should be alterations of the biomineralization system in cholelithiasis: 1) alterations of mucin, 2) alterations in nucleating proteins, and 3) alterations in antinucleating proteins³⁵⁾.

Marked increase in the concentration of cholesterol-nucleating protein has been found in the bile from patients with cholesterol gallstones. This protein, however, is not shown to be increased in concentration in the bile of pigment gallstones. We agree with Strasberg’s groups that there should be an alteration in protein in lithogenic bile, and these proteins may be nucleated cholesterol or calcium bilirubinate from gallbladder bile⁶⁾.

The present result is similar to our previous results which were done with extraction of gallstones³⁸⁾. We still need to revise the extraction method to get more protein.

We try to determine the molecular weight and purity by SDS-PAGE electrophoresis, and the protein bands could be identified by silver staining of the gel. SDS-PAGE showed major bands at approximate molecular weight 66, 45, 29, and 14.2 KDa. The lowest molecular weight bands stained heavily, indicating many protein residues³⁹⁾. Cholesterol gallstones were found to have 14.2 KDa glycoproteins, whereas pigment stones had 66 KDa glycoproteins. All patients with gallstones had 45 KDa glycoproteins, whereas patients without gallstones didn’t. These results suggest that some of these proteins may serve as nucleating agents. The present result differs from the 7 KDa bile-proteins isolated from T-tube bile by Etter-Kjelsaas and Kuenzle⁴⁰⁾. The present study, however, would appear to confirm the result of Shimizu et al. with both experiments suggesting that both cholesterol and pigment gallstones contain acidic proteins with many phosphorylated residues⁴¹⁾. They thus resemble the low molecular weight proteins from normally calcifying tissues (eg. phosphorylations in dental enamels and osteocalain from bone), that regulate the biomineralization processes by binding calcium salts³⁹⁾. They are generally small glycoproteins, which are acidic due to a high content of aspartate, glutamate, phosphorylated/sulfated serine/threonin, and often gamma-carboxyglutamate residues^42,43). Pancreatic and urinary calculi were also found to contain similar proteins^44,45).

We thought that the acidic phosphorylated proteins from gallstones may play a similarly important role in the initiation of nucleation process. However, it remains to be determined whether the protein functions as a promoter or inhibitor of nucleation. Moreover, it is known that the same protein can act as nucleating or antinucleating factors at different concentrations, or under different conditions⁴⁶⁾.

We propose that lithogenic bile may induce an increase in gallbladder protein secretion and gallbladder proteins from both cholesterol and pigment stones could play an important role in the nucleation and growth of the calcium salt crystals. Further studies are necessary to purify and characterize the biliary proteins.