GASTROENTEROLOGY

1992;103:1277-1284

Inhibition of Nucleation and Crystal Growth of Calcium Carbonate by Human Lithostathine JEAN-PAUL BERNARD, ZYGMUNT ADRICH, GIUSEPPE MONTALTO, ALAIN DE CARO, MAX DE REGGI, HENRI SARLES, and JEAN-CHARLES Unit6 de Recherches

DAGORN

de Physiologie

et Pathologie Digestives, INSERM U 315, Marseille, France

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO, crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 pmol/L, could be involved in such a control. Lithostathine in concentrations >l.5 pmol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO, crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO, crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (KJ = 0.9 X lop6mol/L. The glycosylated aminoterminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO, crystal growth with a K,, = 3.0X 10e6mol/L, similar to that af lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO, crystal growth. he ionic composition of pancreatic juice is characterized by high concentrations of calcium and bicarbonate. In fact, those concentrations are such that formation of CaCO, crystals is thermodynamically predictable.’ Yet, crystals are observed in significant numbers in the juice of patients with chronic calcifying pancreatitis only and remain scarce in juice collected from healthy individuals.’ Some sort of control of CaCO, crystal formation or growth must therefore exist in pancreatic secretion, the perturbations of which may eventually result in stone formation. Until recently citrate was the only recognized component of pancreatic secretion able to interact

T

with precipitation of calcium salts because of its properties of chelating divalent cations and possibly by interacting with crystal surfaces. However, preliminary studies suggested that its participation in growth control was limitede3 In other biological fluids supersaturated in a calcium salt, such as saliva, urine and bile, control of crystal growth is partly exerted by small-molecular-weight acidic components such as pyrophosphate and by polyanions such as RNA and glycosaminoglycans4 but also by proteinsa5-* Presence in pancreatic juice of a protein with similar function was suggested when a polypeptide inhibiting in vitro calcium carbonate crystal growth was purified from pancreatic stones.g It was first named pancreatic stone protein (PSP). Further studies showing that PSP found in stones was derived from a secretory protein present in normal pancreatic secretion led us to change its name to lithostathine.” Lithostathine appears in human juice under four forms corresponding to various degrees of glycosylation of the same polypeptide backbone.““’ The peptide bond in position 11-12 of the protein is very sensitive to trypsin cleavage.13 The resulting undecapeptide is hydrophilic and bears the carbohydrate moiety.14 The rest of the protein, formerly called PSP-Sl and now lithostathine H, is hydrophobic and tends to precipitate in the form of polymers as described by Gross et a1.l5 Presence of a protein immunologically related to lithostathine was reported in the juice of five mammals,16 and we showed that the rat protein could inhibit CaCO, crystal growth.16 The present work was aimed at characterizing in vitro the inhibitory properties of human lithostathine and localizing the region of the protein bearing the activity. Materials and Methods Biological Samples creatic

Human disease

pancreatic juice from patients was collected by cannulation

0 1992 by the American

Gastroenterological 0018-5085/92/$3.00

without panof the main Association

1278 BERNARD ET AL.

GASTROENTEROLOGYVol.103,No.4

pancreatic duct during endoscopic retrograde catheterization (J. Sahel, Hapital Sainte Marguerite, Marseille, France). Uncontrolled zymogen activation was prevented during juice collection by adding the following mixture of proteolytic enzyme inhibitors: 2 mmol/L benzamidine, 1 mmol/L phenylmethanesulonyl fluoride, 0.3 mmol/L benzoyl arginine, and 3 mmol/L phenylpropionate. The specificity of lithostathine inhibition of CaCO, precipitation was controlled by using the following proteins: bovine chymotrypsinogen and trypsinogen (Worthington Biochem Corp., Freehold, NJ); porcine colipase and prophospholipase A, (gifts of Dr. R. Verger, Centre de Biochimie et de Biologie Moleculaire, CNRS, Marseille, France); porcine lipase (kindly provided by Dr. M. Rovery, Centre de Biologie Moleculaire, CNRS, Marseille, France); porcine pancreatic polypeptide inhibiting gastrointestinal motility and named pancreatic spasmolytic protein (MR 11698) (Novo Research Institute, Bagsvaerd, Denmark); and human albumin and chicken egg-white lysozyme (Sigma Chemical Co., St. Louis, MO). Purification

of Lithostathine

Native lithostathine forms were isolated according to the immunoaffinity procedures reported previously.” Monoclonal and polyclonal antibodies directed against lithostathine were coupled to AffiGel 10 (Bio-Rad, Richmond, CA). About 40 mg of purified antibodies was immobilized on the affinity columns, and approximately 3.5 mg of pure lithostathine was obtained in one step. Lithostathine H form was obtained by limited tryptic proteolysis of lithostathine, and the glycoundecapeptide generated in these conditions was purified as described previously.‘4 The purity of each peptide preparation was routinely examined by amino acid analysis on a Beckman System 6300 analyzer (Beckman Instruments, San Ramon, CA). It corresponded to the amino acid sequence of the undecapeptide, previously determined as 5-oxoPro Glu Ala Gln Thr Glu Leu Pro Gln Ala Arg. A peptide with identical sequence but without glycan residue on the threonine in position 5 (MR 1532) was synthesized (Neosystemes Laboratoires, Strasbourg, France). In experiments involving hydrolysis of the N-terminal undecapeptide, 1 mg of the natural peptide was incubated with Staphylococcus aureus protease V8 (50 pg; Boehringer Mannheim, Mannheim, Germany) in 0.2 mol/L NH,HCO, (pH 8.1)for 24 hours at 37".Molar concentrations of the polypeptides were estimated on the basis of a molecular weight of 18,000 for lithostathine and a molecular weight of 2000 for the glycosylated N-terminal undecapeptide. Inhibitory Formation

Activity

on CaCO,

Crystal

We used two methods to measure CaCO, precipitation and its inhibition by lithostathine, covering conditions of homogeneous and heterogeneous nucleation. Inhibition of CaCO, crystal nucleation from supersaturated solutions [homogeneous nucleation). CaCO, precipitation experiments were performed according to Wheeler et al.” The course of CaCO, precipitation was monitored under nitrogen atmosphere, by measuring the

decrease in pH consecutive to the rapid mixing of 4 mL of 20 mmol/L CaCl, to 4 mL of 20 mmol/L NaHCO, (pH 8.7). In this model, precipitation of CaCO, initiated by homogeneous nucleation occurred spontaneously after a 2-3-minute delay. The extent of precipitation could have also been estimated from the turbidity of the solution, with similar results’* but less technical convenience. Protein samples were introduced either before CaCl, addition or after the onset of CaCO, precipitation, as indicated in figure legends. A rapid decrease in pH occurred after mixing the solutions, independent of the presence of inhibitors. This has already been reported by other investigators’ and attributed to the formation of stable soluble ion pairsI Inhibition of CaCO, crystal growth (heterogenous nucleation). In this model, the nucleus from which crystal growth will proceed was provided exogenously. This was obtained by seeding a metastable CaCO, solution with weighed amounts of CaCO, crystals.‘9 The seed crystals of CaCO, were prepared by dropwise addition of one volume of 0.2 mol/L NaHCO, to one volume of stirred 0.2 mol/L CaCl, solution at 25°C. The solid phase was separated by decantation and centrifugation, resuspended in ice-cold methanol, centrifuged, and air-dried. The solid phase generated in this system was calcite, as shown by x-ray diffraction. Our seeds were composed of rhombs, each edge measuring 10 pm with a specific surface area of 0.5 m2/g (Coulter Counter TAII Multichannel particle counter; Coultronics SA, Margency, France). The same batch of seed crystals was used in all kinetic studies. Protein samples were added to 20 mL of metastable supersaturated CaCO, solution containing 0.27 mmol/L CaCl, and 4.8 mmol/L NaHCO, adjusted to pH 8.8, under constant stirring. Temperature was maintained at 25°C. In the absence of appropriate nucleating material, the metastable CaCO, solution was stable for 24 hours. Crystal growth proceeded immediately after the addition of 5 mg calcite crystals. Aliquots were removed at various time intervals and filtered through Millipore filters (0.22 pm; Millipore Corp., Bedford, MA), and calcium concentration was measured in the filtrates.” The pH of the solution decreased by 0.15 units during the experiment. This slight decrease was without significant effect on CaCO, precipitation, as judged by comparison with experiments conducted in the presence of 10 mmol/L Tris buffer, pH 8.8; the buffer was therefore omitted. The rates of calcium loss from solution estimated from these data are not strictly quantitative, because supersaturation decreases while precipitation proceeds. However, they provide good approximations suitable for comparative studies describingphysiological situations.5-7 Determination of absolute rates of calcium loss necessary for biophysical studies would require the use of a constant composition crystal growth system based on the principles described for calcium oxalate by Sheehan and Nancollas.21 Adsorption of Lithostathine CaCO, Crystals

and Albumin

on

Adsorption on crystals of lithostathine and human serum albumin was immunodetected by a procedure similar to an enzyme-linked immunosorbent assay (ELISA): a

LITHOSTATHINE

October 19%

specific rabbit antibody reacted with the adsorbed protein and was detected with a goat anti-rabbit antibody coupled with peroxidase. Proteins at a concentration of 5.9 mol/L were incubated for 120 minutes in buffer A (1.5mL of 0.02 mol/L Tris-HCl and 0.150mol/L NaCl, pH 7.5)containing 5 mg CaCO, crystals prepared as described above. Gelatin (0.5%)was included in the buffer when indicated. The supernatant was discarded, and the crystals were washed twice in buffer A. Subsequent treatments were performed in the presence of 0.5% gelatin. The crystals were incubated for 60 minutes with I:2000 dilutions in buffer A of monospecific polyclonal antibodies to lithostathine or albumin. Antibodies to lithostathine were raised in the rabbit using immunologically purified protein as antigen.” Antibodies to albumin were purchased from Nordic Laboratories (Tilburg, The Netherlands). Crystals were rinsed twice in buffer A and then incubated for 60 minutes in a 1:3500dilution in buffer A of goat anti-rabbit IgG antibodies labeled with peroxidase. After three rinses in 0.01 mol/ L Tris-HCl at pH 7.5, peroxidase activity was shown by incubating the crystals with diaminobenzidine in the presence of H,O,. Color development was stopped with 4N H,SO,. The optical density of the supernatant was read at 490 nm in a Kontron (Zurich, Switzerland) Spectrophotometer Uvicon 810/820.All assays were performed in triplicate. Statistica

Linear regressions and tests of significance were performed using standard methods for digital computers (SAS P User’s Guide for Statistics, version 6.03; SAS Institute Inc., Cary, NC). Results on Spontaneous

1279

8.5, \ 7.5 -

‘\

‘-__

I

I

5

----____ I

_______----I

15

I

I

25

Time ( min )

tion before the supersaturated CaCO, solution was formed by CaCl, addition. Lithostathine concentrations (pmol/L) were 1.47 (l), 2.94 (Z), 5.86 (31, 8.82 (4), and 17.6 (5). (B) Lithostathine (8.82 Bmol/L) was added 3 minutes after onset of CaCO, precipitation (arrow). ----, Control CaCO, precipitation in the absence of lithostathine. Experiments were performed at least in triplicate. Data correspond to one representative experiment.

CaCO,

These results are shown in Figure 1. After mixing equimolar amounts of CaCl, and NaHCO,, pH decreased instantaneously from 8.7 to 8.0, then remained relatively stable until nucleation occurred and decreased again as a visible precipitate formed. When lithostathine was added to the bicarbonate solution before CaCl, addition, the duration of the stable period increased in a concentration-dependent manner. Addition of lithostathine after the onset of precipitation diminished the rate at which pH decreased (Figure lB), suggesting a direct inhibitory effect on crystal growth. On the contrary, lithostathine H, and control proteins tested at identical concentrations had no effect. Inhibitory Activity Precipitation

CaCO, PRECIPITATION

Figure 1.Effect of human lithostathine on spontaneous CaC4 precipitation. (A)The protein was added to the bicarbonate solu-

Methods

Inhibitory Activity Precipitation

=

INHIBITS

on Seeded &CO,

Crystal growth from stable supersaturated solutions occurred immediately after inoculation with

calcite seed crystals (Figure 2). Growth was accompanied by a slow decrease in calcium concentration. As shown on Figure 2A, the rate of disappearance of calcium from the solution containing seed crystals was decreased in the presence of lithostathine. The N-terminal undecapeptide of lithostathine and its synthetic analogue showed similar activity (Figures 2B and 3). By contrast, no significant inhibition was observed with lithostathine Hl or various proteins of different size and tissular origin (Figure 4). Control proteins included bovine trypsinogen and chymotrypsinogen, porcine lipase, colipase, phospholipase, and pancreatic spasmolytic protein, human albumin, and chicken egg-white lysozyme. We looked whether the kinetics of calcium loss from solution during precipitation was similar to that reported for other calcium salts” that follow a second order rate law. In that instance, the relation between the rate of CaCO, crystallization from solution and the square of the relative supersaturation should be as follows:

BERNARD ET AL.

GASTROENTEROLOGY

Vol. 103,No. 4

5A) or the N-terminal glycosylated undecapeptide of lithostathine (Figure 5B) did not alter the linearity of the function or the ordinate of the intercept, confirming the validity of Equation 1 for interpreting the experimental results. Quantitative analysis of the effects of polypeptides on CaCO, crystal growth showed a significant decrease of the rate constant (K) with increasing concentrations of the polypeptides (Figure 5A and B). If inhibition is consecutive to the adsorption of these molecules at growth sites on the crystal surface, data should fit in adsorption isotherms6 In simple Langmuir-type adsorption, the relation between the rate constants in the presence

0.25

I1

I

I

1

20 30 40 60 80 TIME (min)

0

I

100

I

120 0.30

h * s

0.25

x

0,25

%

E .3 3 0,20 u 7

0.20

z

0

0,15

I

I

I

I

I

2030 40 60 80 TIME (min)

0

I

I

100

120

2030

40

60 TIME

80

100

120

(min)

4.0 J

B

Figure 2. Influence of increasing concentrations of lithostathine (A) and its N-terminal undecapeptide (B) on the time course of CaCO, crystal growth. Lithostathine concentrations (pmol/L) were 0.6 (l), 1.2(2), 1.8(3), 2.3(4), 2.9(5), and 5.9 (6). Concentrations of undecapeptide (pmol/L) were 1.2(l), 1.8(2), 2.3(3), 2.9 ,and 5.9 (5). Control (0) had no protein added. Data are from (4) one representative experiment of five. Experimental conditions are described under Materials and Methods.

dCa/dt

= -K(Ca, - CaJ,

(Equation I)

where Ca, is the concentration of calcium in solution at any time t, Ca,, is the concentration of calcium in solution at equilibrium (usually 24 hours), and K is a constant rate, the value of which will decrease with inhibition. After integration, Equation 2 is obtained: [Ca, - CaJ’

- [C a, - Ca,,]-’ = Kt,

(Equation 2)

where Ca, is the concentration of calcium at time 0. The integrated rate function (Equation 2) was indeed linear (Figure 5A). Adding lithostathine (Figure

0.0

!

0.00

0.10 (l/[I])xlO

0.20

0.30

-‘ M

Figure 3. Inhibition of CaCO, crystal growth by the synthetic N-terminal undecapeptide of lithostathine. (A) Time course of CaCO, crystal growth in the presence of increasing concentrations of synthetic undecapeptide. Peptide concentrations (pmol/ L) were 1.4(l), 3 (2), 4.5(3), 6 (4), and 9 (5). Crystal growth without peptide (0). Data are representative of one of three experiments. (B) Langmuir adsorption plot for inhibition by the synthetic peptide of CaCO, crystal growth (mean + SE for three experiments). See Figure 6 for legend. Kd was 4.1+ 0.2X 10-' mol/L (R” = 0.936; P < 0.002).

LITHOSTATHINE INHIBITS CaCO, PRECIPITATION

October 1992

1281

background of immunoglobulin binding was nevertheless observed (Table 1). Incubation of 5.9 ymol/L lithostathine with CaCO, crystals resulted in significant binding over background, and addition of gelatin to that incubation only decreased adsorption by 30% (Table 1). Similar results were obtained when human albumin was used in place of lithostathine (Table 1).

0,25

Discussion

0,15(‘.‘.1’.‘.‘..‘.‘..‘.‘.“.“.““,’ 40 2030 0 TIME

We studied in vitro the inhibitory properties of human lithostathine on CaCO, precipitation. The inhibitory activity was localized to the amino-terminal undecapeptide of the molecule. 60

80

100

120

(min)

Figure 4. Influence of other (control) proteins on CaCO, crystal growth. Protein concentration was 6 pmol/L. . . . 0 . . . , Human serum albumin: -Cl--, chymotrypsinogen, lipase, and lysozyme; . - - A . . . , colipase, trypsinogen, and phospholipase A,; . . . ??. . ., pancreatic spasmolytic protein and lithostathineH,; ..+ A..., crystal growth in the absence of exogenous protein. Proteins giving identical results were represented by the same symbol. Experimental conditions are same as in Figure 2.

-7 -g 8

7 -g

8 and absence of inhibitor as follows:

(K, and Kexp, respectively)

10

5

is

K,/(K, - I&,) = 1 + [I]-‘, where I is the concentration of inhibitor. Plots of K,/ (K, - Kexp)vs. l/[I] are shown in Figure 6 for lithostathine and its natural N-terminal undecapeptide and in Figure 3B for the synthetic undecapeptide. The linearity of the relations with an intercept of the ordinate at K,,/(KO - Kexp) near 1 shows that in these experiments, adsorption does follow a Langmuir adsorption isotherm. Values of the dissociation constant (Kd) of the CaCO, crystal inhibitor complex calculated from these plots were 0.9 X lop6 mol/L for lithostathine, 3.0 X lo-’ mol/L for the N-terminal undecapeptide of lithostathine, and 4.1 X 10m6 mol/L for the synthetic undecapeptide. No significant inhibition was obtained with the lowest concentration of synthetic peptide (1.4 umol/L, Figure 3A), and that value was omitted from the least square regression analysis from which the K, was deduced (Figure 3B). Adsorption

of Lithostathine

30

TIME (min)

10

5

to CaCO, Crystals

Gelatin (0.5%) was added to all incubation mixtures containing antibodies, by analogy with ELISA techniques in which omitting saturation of nonspecific protein binding sites with exogenous protein results in high background. In control experiments performed in the absence of lithostathine, a

Figure 5. Calcite crystal growth in the absence and presence of native lithostathine forms (A) and of the N-terminal undecapeptide of lithostathine (B) as expressed by the rate function [Ca, CaJl - [Ca, - CaJ’ vs. time. Figures beside curves refer to the same experimental conditions as in Figure 2.

1282

BERNARD ET AL.

A

GASTROENTEROLOGY Vol. 103,No. 4

3,O

: X:

2.0

@ 0

x

1.0

0.0

0.4 (I/;&o

V,”

1.2 “M

1,6

0.6

' 0.6

I

0.0

0.2

’ 0.4 (lI[I])xlO

-‘ M

Figure 6. Langmuir adsorption plot for inhibition of calcium carbonate crystal growth by lithostathine (A) and its glycosylated N-terminal undecapeptide (B). Values of K, and KsxPrepresent the calcite growth rate constants in the absence and presence of polypeptide (lithostathine or undecapeptide), respectively. [I] represents the concentration of polypeptide. Data are means f SE for five experiments. Kd calculated from ratio of slopes to intercept were 9.9 + 0.92x 10~'mol/L for lithostathine (R’ = 0.936; P = 0.094) and 3.1f 0.3X lo-'mol/L for the undecapeptide(R’ = 0.914; P < 0.001).

Pancreatic secretion is supersaturated in bicarbonate and calcium. Bicarbonate concentrations range from 40 to 120 mmol/L in basal and stimulated secretion, respectively; calcium varies from 0.20 to 0.75 mmol/L in physiological conditions.23s24 Hence, formation of calcium carbonate precipitates is thermodynamically possible. Salt crystallization from supersaturated solutions occurs in two steps. A nucleus with adequate tridimensional structure has to be formed first. Then, further apposition of salt molecules ensures crystal growth. Occasional calcite crystals have been observed in normal juice.’ However, their size is always very small, suggesting that

inhibitors of crystal growth are present in juice. Previous observationsg~16~‘8 pointing at lithostathine as an important element in CaCO, crystal growth control led us to further characterize its interactions with the nucleation and growth of calcite crystals. The experimental conditions chosen for studying nucleation addressed homogeneous nucleation, which is the formation of a crystal nucleus in the absence of interfering factors. This is a simplification of conditions actually occurring in juice, where many components with nonspecific affinity for salts such as proteins and polysaccharides facilitate nucleation by lowering the amount of energy required to initiate the process (heterogeneous nucleation). Lithostathine, contrary to other secretory proteins, decreased the rate of homogeneous nucleation of calcite (Figure l), which shows that the protein is already interfering with the very first steps of lithogenesis. Influence of lithostathine on CaCO, crystal growth, the second step of lithogenesis, was then studied by seeding a supersaturated solution with preformed crystals. That model is a good approximation of what occurs in physiological conditions. We could show that inhibition of growth was a function of lithostathine concentration. Also, calculation of a Langmuir isotherm for that reaction showed that the protein was specifically adsorbed to crystal growth sites, with a K, of 0.9 X 10m6 mol/L. Again, none of the proteins tested as control displayed similar properties, suggesting that control of CaCO, crystal growth may be the major physiological role of lithostathine. Localizing the region of lithostathine involved in inhibition implied that fragments of the protein could be purified and individually tested. Mild trypsin hydrolysis of the protein generates two peptides of 11 and 133 amino acids.14 The amino-terminal undecapeptide is highly soluble and bears the sugar moiety of the protein. The carboxy-terminal peptide has a strong tendency to polymerize between pH 6 and pH 9 in the form of insoluble fibrils.” The limited amounts remaining in solution did not show inhibitory activity. On the contrary, the undecapeptide had retained the inhibitory properties of lithostathine, with a similar K, for CaCO, crystals (3.0 X lo-’ mol/L). Abolition of inhibition after treating the peptide with protease V8, which generates three fragments, suggested that the glycan chain was not responsible by itself for the inhibition. Actually, it might not be involved at all, because a synthetic peptide with the sequence of the undecapeptide but without glycan was equally active, with a K, of 4.1 X lop6 mol/L (Figure 3B). None of the proteins tested in this study including

LITHOSTATHINE

October 1992

Table 1. Adsorption of Lithostathine

INHIBITS CaCO, PRECIPITATION

and Albumin to Calcium Carbonate Crystals Albumin

Lithostathine 1 Protein Antibody 1 Absorbance (xl@ f SE)

1283

2

3

4

5

6

7

8

None a litho

Gelatin a litho

Litho a litho

Gel + Litho a litho

None a alb

Gelatin a alb

Albumin a alb

Alb + gel a alb

340 f 21

364 f 13

725 f 31

618 + 24

530 + 12

560 + 15

1110 + 9

1460 + 22

NOTE. Lithostathine and human serum albumin were used (5.9 X lo-'mol/L). Polyclonal antibody to lithostathine was diluted 1:2000. Polyclonal antibody to human serum albumin was diluted 1:ZOOO.Second antibody is a goat anti-rabbit polyclonal antibody, peroxidase labeled (dilution, 1_3500). Absorbance was read at 490 mm. Litho, lithostathine; gel, gelatin; alb, albumin.

albumin could significantly inhibit CaCO, precipitation, except lithostathine (Figure 4). Yet, albumin showed a strong binding to CaCO, crystals, as already shown for crystals of other calcium salts,” and it is likely that other proteins devoid of inhibitory activity on crystal growth equally bind to calcite. Hence, lithostathine appears as the only tested protein whose binding on calcite crystals will interfere with growth sites, thereby preventing further apposition of calcium carbonate. Proteins similar to human lithostathine in their structure and inhibitory activity have been described in other mammals. In dog, a protein with a molecular weight (MR) of 15,000 that immunologically related to lithostathine was shown to inhibit CaCO, crystal nucleation.‘” In rat, a protein with a M, of 16,000 inhibiting nucleation and crystal growth with a K, of 1.5 X 10d6 mol/L was purified from juice.16,25 Its sequence deduced from the nucleotide sequence of its mRNA showed 75% similarity with human lithostathine. Immunologically related proteins have also been found in pancreatic secretion from cow, swine, and monkey,‘6J6 suggesting that presence of proteins controlling crystal growth is required in mammalian juice. Similar observations have been made in other biological fluids in which calcium salt precipitation would occur in the absence of inhibitors, such as saliva5f7 and urine.6’27 The inhibitors described in these secretions, proline-rich proteins and statherine in saliva5p7 and nephrocalcine in urine,6 have in common with lithostathine a low molecular weight and an acidic p1 due to a high content in dicarboxylic acids. Their inhibitory properties have been tentatively attributed to the presence of phosphorylated residues’ or 27both resulting from posttransy-carboxyglutamate, lational maturation. However, neither could be evidenced in lithostathine. K, of proline-rich protein or statherine for calcium phosphate has not been determined. The K, of nephrocalcine for calcium oxalate is 1.9 X 10-s mol/L, two orders of magnitude higher than the K, of lithostathine for CaCO,. This may re-

flect the low concentration of nephrocalcine in urine, compared with lithostathine in juice. Association between structural alterations of the inhibitor and lithiasis was established for nephrocaltine.” The content in y-carboxyglutamate of the protein purified from patients with lithiasis was significantly lower, resulting in a decreased inhibitory activity of the protein. Preliminary studies conducted on lithostathine purified from patients who had chronic calcifying pancreatitis could not evidence structural changes, and the K, of the protein for CaCO, crystals was not altered. It is known, however, that lithostathine concentration is diminished in the juice of patients” and that its messenger RNA concentration is decreased in pathological pancreas.” Contrary to urinary lithiasis, pancreatic lithiasis may be related to insufficient inhibition of crystal growth as a result of abnormal lithostathine gene expression. References 1. Moore EW, Verine H. Pathogenesis

2.

3.

4.

5.

6.

7.

of pancreatic and biliary CaCO, lithiasis: the solubility product (K’sp) of calcite determined with the Ca++ electrode. J Lab Clin Med 1985;106:611618. Multigner L, Mariani A, Sahel J, De Caro A, Sarles H. Occurrence of calcium carbonate crystals in normal and pathologic pancreatic Juice. Digestion 1987;38:44-45. Boustiere C, Sarles H, Lohse J, Durbec JP, Sahel J. Citrate and calcium secretion in the pure human pancreatic juice of alcoholic and nonalcoholic men and of chronic pancreatitis patients. Digestion 1985;32:1-19. Ryall RL, Harnett HM, Marshall VR. The effect of urine, pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1981;112:349-356. Hay DL, Moreno EC, Schlesinger DH. Phosphoproteins inhibitors of calcium phosphate precipitation from salivary secretions. Inorg Persp Biol Med 1979;2:271-285. Nakagawa Y, Abram V, Kezdy FJ, Kaiser ET, Coe FL. Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. J Biol Chem 1983;258:12594-12600. Schlesinger DH, Hay DI. Complete covalent structure of statherine, a tyrosine rich acidic peptide which inhibits calcium

1284 BERNARD ET AL.

phosphate precipitation from human parotid saliva. J Biol Chem 1977;252:1689-1695. 8. Shimizu S, Sabsay B, Veis A, Ostrow JD, Rege RV, Dawes LG. Isolation of an acidic protein from cholesterol gallstones which inhibits the precipitation of calcium carbonate in vitro. J Clin Invest 1989;84:1990-1996. 9 De Caro A, Lohse J, Sarles H. Characterization of a protein isolated from pancreatic calculi of men suffering from chronic calcifying pancreatitis. Biochem Biophys Res Commun 1979;87:1176-1182. 10.Sarles H, Dagorn JC, Giorgi D, Bernard JP. Renaming pancreatic stone protein as “lithostathine.” Gastroenterology 1990;99:900-901. 11.Montalto G, Bonicel J, Multigner L, Rovery M, Sarles H, De Caro A. Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein. Biochem J 1986;238:227-32. 12. De Caro A, Bonicel J, Rouimi P, De Caro J, Sarles H, Rovery M. Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice. Eur J Biochem 1987;168:201-207. 13. Rouimi P, Bonicel J, Rovery M, De Caro A. Cleavage of the Arg-Be bond in the native polypeptide chain of human pancreatic stone protein. FEBS Lett 1987;216:195-199. 14. De Caro A, Adrich Z, Fournet B, Capon C, Bonicel J, De Caro J. Rovery M. N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5 oxoproline N-terminal chain is 0 glycosylated on the 5th amino acid residue. Biochem Biophys Acta 1989;994:281-284. 15. Gross JR, Carlson RI, Brauer AW, Margolies MN, Warshaw AL, Wands JF. Isolation, characterization and distribution of an unusual pancreatic human secretory protein. J Clin Invest 1985;76:2115-2126. 16. Bernard JP, Adrich Z, Montalto G, Multigner L, Dagorn JC, Sarles H, De Caro A. Immunoreactive forms of pancreatic stone protein in 6 mammalian species. Pancreas 1991;6:162167. 17. Wheeler AP, Georges JW, Evans CA. Control of calcium carbonate nucleation and crystal growth by soluble matrix of oyster shell. Science 1981;212:1397-1398. 16. Multigner L, De Caro A, Lombard0 D, Campese D, Sarles H. Pancreatic stone protein, a phosphoprotein which inhibits calcium carbonate precipitation from human pancreatic juice. Biochem Biophys Res Commun 1983;110:69-74. 19. Reddy MM, Gaillard WD. Kinetics of calcium carbonate seed crystallization, Influence of solid solution ratio on the reaction constant. J Co11 Interface Sci 1981;80:171-178.

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Received September 26, 1991. Accepted April 22,1992. Address requests for reprints to: Jean-Charles Dagorn, M.D., U 315 INSERM, 46 Boulevard de la Gaye, 13009 Marseille, France. Supported by grant no. 639/87 from the Caisse Regionale d’Assurance Maladie des Travailleurs du Sud-Est. Z. Adrich was supported by a fellowship from the Fondation pour la Recherche Medicale (Paris). The authors thank A. Guidoni for performing amino acid analyses. Part of this work was presented at the annual meeting of the American Gastroenterological Association (San Antonio, 1990) and was published in abstract form (Gastroenterology 1990; 98:A214.).