Proc. NatI. Acad. Sci. USAVol. 75, No. 12, pp. 6230-6234, December 1978Medical Sciences

Cholesterol monohydrate growth in model-bile solutions(cholelithiasis/crystal morphology/diumal variation/solubility)

E. W. TOOR, D. F. EVANS, AND E. L. CUSSLERDepartment of Chemical Engineering, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213

Communicated by Clarence Zener, September 5, 1978

ABSTRACT The growth of cholesterol monohydrate fromsolutions of bile salt,lecithin, and small electrolytes has beenstudied by microscopy and with the Coulter Counter. The crystalforms found by micocopy are the same as those seen in humangallstones and in squirrel monkey cholesterol microliths. Thecholesterol growth rates determined with the Coulter Countervary slowly with cholesterol concentration at low degree ofsupersaturation but become exponential at higher cholesterolconcentrations. Growth is accelerated by the presence of cal-cium and magnesium but inhibited by potassium. These resultscan be combined with previous measurements of cholesteroldissolution rates to give a more accurate picture of the dynamicsof gallstone formation.

The complex nature of real bile, in which many factors maychange from sample to sample, makes it difficult to study nu-cleation and growth systematically. The use of model bile so-lutions allows the study of growth under controlled conditions.Therefore, we have studied the growth rates of cholesterolmonohydrate from model bile solutions containing sodiumtaurocholate, lecithin, cholesterol, and electrolytes and frommixtures of human and model bile, using the Coulter Counterand polarizing microscope.

Such studies can clarify the causes of cholesterol gallstoneformation in several ways. First, observation of the morphologyof cholesterol monohydrate crystals grown in model solutionscan show whether other components of real bile are needed toproduce the crystal forms found in gallstones. Second, it can bedetermined whether the rate of growth of cholesterol in modelsystems is comparable to the few existing observations of gall-stone growth rates in vivo. Third, if the rates and mechanismsof growth in model solutions are known, it becomes possible totest the effects of other components of bile, one by one, and tosearch for inhibitors and promoters of growth. Finally, growthrates can be combined with previously measured dissolutionrates to study the effects of diurnal variation of gallbladder bilecholesterol concentration on stone formation. Information aboutthe nucleation process derived from these measurements willbe discussed elsewhere (1).

EXPERIMENTALModel bile solutions were made by coprecipitation (2) of sodiumtaurocholate, lecithin, and cholesterol. Grade A sodium tau-rocholate (Calbiochem) was recrystallized by the method ofPope (3) and dissolved in methanol; the solution was passedthrough a Sephadex LH-20 column and evaporated to dryness.We found that this procedure was necessary to remove the NaClpresent in sodium taurocholate as received and to removesurface-active impurities. Surface tension was measured overa range of concentrations with a DuNuoy tensiometer. A re-producible surface tension curve without a dip at the criticalmicelle concentration could only be obtained if both recrys-tallization and purification on the column were done. Grade

A lecithin (Lipid Products) was used; and reagent grade cho-lesterol (Fisher) was recrystallized from benzene or ethanol.Purified sodium taurocholate and lecithin and coprecipitatedsamples were stored at -50C.To make coprecipitated samples, appropriate amounts of

sodium taurocholate, lecithin, and cholesterol were dissolvedin a mixture of chloroform and methanol, mixed, evaporatedunder reduced pressure, and dried under vacuum for ap-proximately 48 hr, until the weight loss in 12 hr was less than0.1% of the total sample weight. Unless vacuum drying is pro-longed, traces of methanol are found by using 250-MHz NMRwhich resolves the methanol methyl proton peaks from the(CH3)3 N peaks in lecithin. All samples for the Coulter Counterwere made up with sodium taurocholate/lecithin/cholesterolmolar ratios of 8:2:X, with the cholesterol molar ratio X varyingfrom 0.34 to 0.79.

Electrolyte solutions were made up from Analyzed ReagentNaCl or CaCl2 (Baker) in water from a Stokes still. For theCoulter Counter, all solutions were 0.15 N and were filteredthrough 0.22-tim Millipore filters to achieve negligible back-ground particle count and to remove microorganisms. Sufficientelectrolyte solution was added to a coprecipitated sample tomake it 10% by weight in total lipids. The sample was thensupersaturated by heating in a water bath at approximately50'C for 1 hr; any undissolved cholesterol was removed byfiltering the warm suspension through a 0.22-;im Milliporefilter. The resulting clear sterile solution was placed in theCoulter Counter and allowed to come to room temperature (23i 10C). The time at which room temperature was reached(approximately 2 hr) was taken as zero for Coulter Countermeasurements. Samples were sealed except during CoulterCounter runs.The cholesterol concentration in the supersaturated solution

was determined by subtracting the amount of cholesterol lefton the Millipore filter from the total amount added to theoriginal sample. The filter through which the heated super-saturated solution had been passed was washed with 5 ml ofwater to remove any residual sodium taurocholate or lecithin,dried, and then extracted with chloroform to dissolve the cho-lesterol. The chloroform solution was evaporated to dryness andthe remaining cholesterol was determined by weight. As a checkon this procedure, a mixture (8:2:0.60 mole ratio) containingradioactive [14C]cholesterol (New England Nuclear) was pre-pared as above and the amount of cholesterol retained on thefilter was measured both gravimetrically and by scintillationcounting. Duplicate determinations by each method differedby less than 0.05% of the total cholesterol in the sample. Stainlesssteel filtration systems were used because we found evidenceof adsorption of cholesterol on glass as observed by Haberlandand Reynolds (4). We found no evidence of cholesterol ad-sorption on stainless steel.

Particle size distributions were measured with a CoulterCounter model TA II (5, 6) using either a 30-.um aperture tube(particle diameter range, 0.6-12 Mm) or a 100-tm aperture tube(particle diameter range, 2-40 Mm). Measurements were madeat intervals of a few hours to a day, depending on the growth

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rate of the sample, and were continued for up to a month. Theparticle number versus diameter distributions from the counterwere converted to logarithmic normal distribution-plots to findthe median particle size as a function of time (7). Samples formicroscopic observation were removed during Coulter Counterruns to determine what crystal forms were present. Duplicateruns in the low supersaturation range gave almost identicalgrowth rates; at higher degrees of supersaturation, particularlyaround the transition point found in the growth rate curve, thescatter was much larger.

In a parallel series of experiments, samples of human T-tubebile were mixed with a highly supersaturated model bile solu-tion and the subsequent crystal growth was followed with theCoulter Counter. Human bile samples were filtered througha coarse fritted glass filter and through 1.2- and 0.22-/um Mil-lipore filters to remove suspended material which would in-terfere with the Coulter Counter measurements. The growthrate of cholesterol particles in the highly supersaturated solutionwas determined before addition of human bile.

Samples for microscopy were sealed in capillary microslides(Vitro Dynamics) with an optical path length of 0.22 mm andobserved with a Nikon polarizing microscope at room tem-perature or at 370C. In addition to slides made from CoulterCounter solutions, a number of samples were made for mi-croscopy only, with variation in the electrolyte used or the so-dium taurocholate-to-lecithin ratio.

RESULTS AND DISCUSSIONGrowth Habit. As supersaturation was increased, the ap-

pearance of crystals grown from model bile solutions changed.The basic form was a very thin pseudorhombohedral plate. Theslowest growth direction was perpendicular to the plate face.

A SOf..~~~~~~~~~~~~Proc. Nati. Acad. Scd. USA 75 (1978) 6231

At low supersaturations, only these plates were seen. At highersupersaturations, spiral dislocation growth patterns occurred(Fig. IA), followed by both dislocation spirals and occasionaltwin crystals (Fig. 1B). Eventually twinning became dominant(Fig. 1C). Finally, at cholesterol concentrations at which thesolution became cloudy within a few minutes, there was anamorphous precipitate (nonbirefringent and apparently notliquid crystalline) that gradually was replaced by very largeplates heavily twinned at the edges (Fig iD).The forms seen were the same in 0.075 M NaCl, 0.15 M

NaCl, or 0.30 M NaCl, although crystals appeared faster inhigher concentrations of electrolyte. However, when the solventcontained 0.075 M CaCl2, new crystal faces appeared, modi-fying the plate shape so that plates had pointed ends or weresix-sided. At high cholesterol concentrations, clumps of ra-diating crystals occurred (Fig. 1 E and F). MgCl2 had similarbut smaller effects. On the other hand, in 0.15 M KCI, nocrystals were found after 15 days at a cholesterol concentrationat which crystals appeared in 1 day in NaCl. The same crystalforms grew with or without lecithin (Fig. 1E). We have alsogrown cholesterol monohydrate crystals from ethanol/watersolutions, and these were similar in form to those grown frommodel bile solutions with high cholesterol supersaturation.The similarity of the crystal forms observed under these

widely varying conditions is evidence that the growth mecha-nisms are characteristic of cholesterol monohydrate and not ofthe model bile solutions. These crystal forms strongly resemblethe microliths observed in squirrel monkeys (8). The projectingtwins found at high supersaturation and the radiating clustersfound in calcium solutions resemble the projections often seenin cross sections of human gallstones (9).Growth Rates. The particle size distribution curves found

for cholesterol monohydrate crystals.are logarithmic normal

C

A,~~~ ~ ~, '

v~ v

FIG. 1. Cholesterol microcrystals. (A-D) Sodium taurocholate/lecithin 8:2; cholesterol mole ratio increasing progressively from approximately0.55 to 0.80; 10% by weight of 0.15M NaCl. Because the cholesterol removed by filtration of solutions was not measured, exact cholesterol con-centrations are not known. (E) Sodium taurocholate/lecithin/cholesterol, 98:0:2; 10% by weight in 0.075 M CaCl2. (F) Mixture of-model bile(sodium taurocholateAecithin/cholesterot, 8:2:0.72; 10% by weight in 0.15 M NaCl) and human T-tube bile. The bar in each picture represents25 Mm.

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a

£3

Ca2C

0 2Time (hr x10-2)

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FIG. 2. Mean particle diameter versus time (in 0.15 M NaCIat 230C). Sodium taurocholate/lecithin/cholesterol ratios; 0, 8:2:0.40;A, 8:2:0.50; 0, 8:2:0.72.

and so yield the mean geometric sizeM plotted versus time inFig. 2. Before growth could be measured, there was an induc-tion period that was longer at low cholesterol concentrations:for example, for the ratio 8:2:0.40 in Fig. 2 the induction timewas 170 hr. After the induction period, the mean size increasedlinearly with time. As the supersaturation was decreased bygrowth, growth slowed. The linear growth rate, R, was mea-

sured when the amount of solid formed was less than 1% of thetotal amount of cholesterol in solution. Typical growth rates are

given in Table 1 and plotted versus cholesterol concentrationin Fig. 3.The growth rates for cholesterol are much smaller than those

usually found for ionic crystals (10). This is apparently a char-acteristic not of the model bile salt solutions but of cholesterolmonohydrate itself, which grows in 0.15 M NaCl at about 0.05Aum/hr (11), in the same range as that found here in model bilesolutions. Why growth rates are faster in solutions containingCa2+ and Mg2+ is unclear.To find the supersaturation of these solutions, the solubility

of cholesterol must be known. The solubility can be determined

Table 1. Typical growth rates

Cholesterol, Degree of Growth rate,In mole ratio* saturationt jim/hr

0.15 M NaCl 0.38 0.15 0.00170.40 0.21 0.0030.40 0.21 0.0030.44 0.33 0.0130.50 0.51 0.0250.50 0.51 0.0270.60 0.82 0.0400.69 1.09 0.1150.70 1.12 0.0510.72 1.18 0.0760.78 1.35 0.3250.79 1.39 1.30

0.075 M CaCl2 035 0.06 0.0080.47 0.42 0.530.70 1.12 0.75

0.075 M NaCl + 0.50 0.52 0.130.0375 M CaCl2 0.62 0.88 0.40

At 230C; total lipids, 10% (wt/vol).* In sodium taurocholate/lecithin, 8:2.t Given by (concentration - solubility)/solubility.

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0.20 0.40 0.60 0.80 1.00

Mole RatioFIG. 3. Growth rate and observed crystal form versus cholesterol

concentration (mole ratio in sodium taurocholate/lecithin, 8:2) at230C. 0, In 0.15M NaCl; A, in 0.075M CaCl2; D, in 0.075M NaCl plus0.0375 M CaCl2.

by extrapolating the Coulter Counter measurements shown inFig. 3 to find the concentration at which the growth rate is zero;the value of 0.33 mole ratio of cholesterol is obtained in NaCland CaCl2 solution.The variation of growth rate with degree of supersaturation,

S, thus obtained can be explained by two types of crystal growththeory. The first, the Burton-Carbrera-Frank screw dislocationmodel (12) assumes dislocations (stacking faults in the crystal)which provide a self-perpetuating spiral ledge on the surfacealong which growth outward can occur continuously. Thismodel predicts that, at low supersaturation, the growth rate willbe proportional to S2 but at higher supersaturation the growthrate changes to become proportional to S.

However, crystal growth occasionally shows a still sharperdependence on supersaturation. Such a dependence is explainedby the surface nucleation model (13) which assumes that mol-ecules adsorb upon the crystal surface and migrate across it toform two-dimensional surface nuclei. Additional adsorbedmolecules migrate to the nucleus which grows outward across

the surface to complete a crystal layer. When the rates of nu-

cleus formation and surface spreading are comparable, thegrowth rate varies exponentially with the degree of supersat-uration.

In Fig. 4 the logarithm of the growth rate is plotted as a

function of log S for all samples. For model solutions containingNaCl, the data lie on a curve with a break at S = 1.1, or sodiumtaurocholate/lecithin/cholesterol molar ratio of 8:2:0.68. BelowS = 1.1, the data fit that range of the screw dislocation modelin which the slope changes from 2 to 1. This is the region inwhich we see predominantly screw dislocations under the mi-croscope. Above S = 1.1, growth becomes exponential, fittingthe surface nucleation model. This is the region where twinningbecomes dominant.The visual similarity of form between cholesterol microliths

grown in vitro and in vivo is reinforced by the growth experi-ments involving mixtures of human T-tube bile and model bile.Four different filtered human bile samples were used, eachmixed in several different proportions with a concentratedmodel solution. For three of the human bile samples, the formof twinned crystals already present in the model solution didnot change, but the number per unit volume and the growth

-I /, __

I/II

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Proc. Natl. Acad. Sci. USA 75 (1978) 6233

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Degree of Supersaturation

FIG. 4. Growth rate as a function of degree of cholesterol super-saturation (S) in sodium taurocholate/lecithin (8:2) at 230C. 0, In0.15 M NaCl; A, in 0.075 M CaCl2; 0, in 0.075 M NaCl plus 0.0375 MCaCl2-

rate decreased in proportion to the relative amounts of real andmodel bile. For the fourth human bile sample, the preexistingcrystals began to show the forms and increased growth ratefound in our CaCI2 solutions.The growth rates of cholesterol crystals in model solutions

are of the same order of magnitude as the growth rates ofgallstones in vivo. Observed growth rates are consistent withthe report by Wolpers and Blaschke (14) that a faceted singlestone took about 8 years to grow, whereas aggregated radiatingcluster stones took about 2 years to grow. It is difficult to makequantitative comparisons because the morphology of gallstonesreflects the highest cholesterol concentration encountered butthe growth rate depends upon average cholesterol concentrationduring the growth period. Our rate data agree closely with thetimes required to grow squirrel monkey microliths (8). Althoughother biliary components such as bilirubin or mucoprotein arenot necessary to explain the crystal morphology or growth ratesobserved, such components may affect nucleation or providea mechanism for microlith retention over many cycles of gall-bladder function.

Cholesterol Solubility. The solubility of cholesterol in thesodium taurocholate/lecithin (8:2) solution as determined byCoulter Counter measurements was 0.33 mole ratio (or 5.6mM), considerably lower than the values reported by otherinvestigators (15-19). Because the solubility of cholesterol in

bile is crucial in deciding when gallstones will grow or dissolve,the reasons for this discrepancy must be examined.The previous studies used a common method: the bile salt,

lecithin, and cholesterol solutions were filtered through Milli-pore filters or centrifuged prior to analysis. When the filtrationtechnique was used in our laboratory to determine the apparentsolubility of cholesterol (20), solubilities in the range found byother investigators were obtained. Thus, the discrepancy is notcaused by differences in sample preparation among investi-gators but by the difference in method of measuring micellarsolubility.With the Coulter Counter, the solubility of cholesterol is

determined by finding that cholesterol concentration at whichno growth occurs (Fig. 3). The lower solubility obtained in thisway is also substantiated by microscopy. With sodium taur-cholate/lecithin/cholesterol at 8:2:0.50, very small crystalliteswere formed after 2 weeks of growth. When the cholesterolconcentration was lowered to 8:2:0.44, crystals became visibleunder the microscope after 1 month; these crystals were so smallthat they might have been missed if the Coulter Counter resultshad not suggested that growth was occurring. At 8:2:0.40, nocrystals could be seen at a magnification of X200 after 5 weeks,although the Coulter Counter indicated a slow growth to a di-mension of about 1 Atm. At low supersaturation, induction timescan be so long and growth so slow that only a fraction of thecholesterol in these solutions has precipitated after severalweeks.

At high cholesterol supersaturations (>0.69 mole ratio), thegrowth rate increases exponentially with concentration (Figs.3 and 4) and rapid formation of gallstones can occur. This valuelies below the empirical metastable/labile cholesterol con-

centration of about 8:2:1.1 mole ratio determined by visualinspection of a solution within minutes after preparation (19,21). However, nucleation and growth rates that result in im-mediate visible cloudiness must be orders of magnitude greaterthan those at which the onset of exponential growth is observedwith the Coulter Counter, in which case several hours are re-

quired to produce visible crystals.By microscopy, solutions with cholesterol concentrations high

enough to produce cloudiness to the eye in seconds or minutesafter filtration contained not crystalline cholesterol but an

amorphous nonbirefringent material that gradually disap-peared and was replaced by large, heavily twinned cholesterolcrystals. The amorphous material may be a nonordered mixtureof submicroscopic cholesterol crystals and lecithin that gradu-ally rearranges to large cholesterol crystals and lecithin in mixedmicelles. These results suggest that, in vivo, cholesterol con-centrations at which growth can begin and at which it can be-come rapid are considerably lower than previously believed.Thus, the kinetic factors involved in gallstone formation anddissolution must be reexamined.

Table 2. Calculated growth and dissolution during diurnal variation

Time atStone Ratio: subsaturation 24-hrdiam., Growth dissolution rate for no average

Fluctuation* cm ratet growth rate net growth saturation

+10%, -10% 0.1 1.4 500 3 min 1.11 1.4 250 6 min 1.1

+50%, -10% 0.1 36 20 1.1 hr 1.51 36 10 2.2 hr 1.4

+100%, -10% 0.1 70 10 2.2 hr 1.91 70 5 4.0hr 1.8

+150%, -10% 0.1 2780 0.25 19 hr 1.21 2780 0.125 21 hr 1.1

* Shown as above and below cholesterol saturation.t Expressed here as gcm-2-sec-1 X 1011; increase of particle mass per unit area with time.

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Physiological Conditions for Gallstone Growth. As a resultof the enterohepatic circulation, the composition of bile variescontinuously during the day. During fasting, cholesterol con-centration increases; during feeding it decreases (22). Thus,gallbladder bile can be periodically supersaturated and less thansaturated, and gallstones can alternately grow and dissolveduring a 24-hr period. The net effect depends upon the relativerates of growth and dissolution while the bile is supersaturatedor subsaturated.By combining the cholesterol growth rates with previously

measured dissolution rates (23), the effects of the diurnalvariation in cholesterol concentration can be explicitly con-sidered. As an example, consider the conditions (Table 2) inwhich the amount of cholesterol gained during growth justequals the amount lost by dissolution during a 24-hr period,resulting in no net growth. Previously, we have measured dis-solution as a function of a wide variety of physical and chemicalfactors. Unfortunately, the values of these factors in vvo areunknown, particularly the bile flows that are associated withfilling, emptying, and solute concentration in the gallbladder.To be conservative, we choose these factors as those associatedwith free convection. This convective flow will always arisefrom the density differences caused by the cholesterol con-centration gradient from the stone surface to the bulk solution.Under these conditions, dissolution is slower than suggested bymany earlier measurements (24). In sodium taurocholate/lec-ithin/cholesterol at 10:30:0, this slower value is 7 X 10-8 g-cm 2sec-1 for a 0.1-cm-diameter stone and 3.5 X 10-8 g*cm-2-sec-1 for a 1.0-cm stone (21). As cholesterol concentrationis increased, the dissolution rate will decrease linearly and ap-proach zero at saturation.When these dissolution rates are combined with our growth

rates, we can estimate how long each day the bile must besubsaturated in order to produce no net growth. Typical resultsare shown in Table 2. For small fluctuations above and belowsaturation, growth is much' slower than dissolution (rows 1 and2). Thus, long periods of supersaturated bile can be tolerated(column S) without any net 'growth occurring, even though thedaily average concentration of cholesterol in the gallbladdercan be above saturation (column 6). As the size of the fluctua-tions above saturation increase (rows 3 and 4), the bile mustremain subsaturated for longer periods of time to prevent netgrowth. This happens because the dissolution rate dependslinearly on cholesterol concentration but the growth rate ap-proaches an exponential dependence at high cholesterol su-persaturation.The analysis leading to Table 2 is approximate in several

ways. It implies abrupt concentration changes of well-mixedgallbladder bile. In fact, these changes will be gradual andmixing will be incomplete. These differences will modify theanalysis given in Table 2 but not the qualitative conclusions.Furthermore, the induction times we find in model solutionssuggest that there may be additional protection against initiallyforming cholesterol nuclei at low supersaturation. It is not yetknown how well induction times in model solutions approxi-mate those in human bile, but there is some evidence that in-duction times may be considerably greater in vio than in vitro(25).

Thus, in spite of the low solubility of cholesterol there is alarge range of physiologically significant conditions underwhich there will be no formation or no net growth of gallstones.At low supersaturation, dissolution is faster than growth so thatany material precipitated while bile is supersaturated canrapidly dissolve when bile becomes subsaturated.

CONCLUSIONS(i) Cholesterol precipitates from solutions containing bile

salt, lecithin, and small electrolytes show the same crystal formsas those observed in real bile, both in vitro and in vivo.

(ii) Cholesterol monohydrate growth rates in vitro arecomparable to growth rates in vivo.

(iii) Calcium and magnesium ions promote growth but po-tassium ions inhibit either nucleation or growth. A basis ofcomparison has been established for testing other possible in-hibitors or promoters.

(iv) The solubility of cholesterol found from growth ratestudies is lower than previously believed at the bile salt/lecithinratio studied.

(v) Growth and dissolution rates can be combined to ap-proximate the effects of the diurnal variation in cholesterolconcentration on stone growth.We thank Dr. H.; Fromm (Gastroenterology Department, Montefiore

Hospital, Pittsburgh, PA) for supplying human bile samples and W.J. Benton (Department of Chemical Engineering, Carnegie-MellonUniversity, Pittsburgh, PA) for photomicrography. This work wassupported by National Institute of Arthritis, Metabolism, and DigestiveDiseases Grant AM-16143.

1. Toor, E. W., Evans, D. F. & Cussler, E. L. (1979) in Hepatol-ogy-Research and Clinical Issues, ed. Fisher, M. M. (Plenum,New York), Vol. 4, in press.

2. Small, D. M., Bourges, M. C. & Dervichian, D. G. (1966) Biochim.Biophys. Acta 125,563-580.

3. Pope J. L. (1967) J. Lipid Res. 8, 146-147.4. Haberland, M. E. & Reynolds, J. A. (1973) Proc. Nati. Acad. Set.

USA 70,2313-2316.5. Allen, T. (1968) Particle Size Measurement (Chapman and Hall,

London), pp. 143-153.6. Batch, B. A. (1964) J. Inst. Fuel 37,455-461.7. Irani, R. R. & Callis, C. F. (1963) Particle Size: Measurement,

Interpretation and Application (Wiley and Sons, New York),pp. 34-57.

8. Portman, 0. W., Osuga, T. & Tanaka, N. (1975) Adv. Lipid Res.13, 1M5-94.

9. Rains, A. J. H. (1964) Gallstones (Thomas, Springfield, IL), p.16.

10.- Ohara, M. & Reid, R. C. (1973) Modelling Crystal Growth Ratesfrom Solution (Prentice-Hall, Englewood Cliffs, NJ), pp.113-134.

11. Higuchi, W. I. & Saad, H. Y. (1965) J. Pharm. Sci. 54,72-78.12. Burton, W. K., Cabrera, N. & Frank, F. C. (1951) Philos. Trans.

R. Soc. London Ser. A 243, 299-8.13. Ohara, M. & Reid, R. C. (1973) Modelling Crystal Growth Rates

from Solution (Prentice-Hall, Englewood Cliffs, NJ), pp. 19-45.

14. Wolpers, C. & Blaschke, R. (1971) Proc. Electron Microsc. Soc.Am. 29, 296-297.

15. Neiderheiser, D. H. & Roth, H. P. (1968) Proc. Soc. Exp. Biol.Med. 128, 221-225.

16. Hegardt, F. G. & Dam, H. (1971) Z. Ernaehrungswiss. 10,223-233.

17. Holzbach, R. T., Marsh, M., Olszewski, M. & Holan, K. (1973)J. Clin. Invest. 52,1467-1479.

18. Mufson, D., Triyanond, K., Zarembo, J. E. & Ravih, L. J. (1974)J. Pharm. Sci. 63,327-32.

19. Carey, M. C. & Small, D. M. (1978) J. Clin. Invest. 61, 998-1026.

20. Shaeiwitz, J. A. (1978) Dissertation (Carnegie-Mellon University,Pittsburgh, PA).

21. Admirand, W. H. & Small, D. M. (1968) J. Clin. Invest. 47,1043-1052.

22. Grundy, S. M. (1973) Hosp. Pract. 8,57-65.23. Tao, J. C., Cussler, E. L. & Evans, D. F. (1974) Proc. Natl. Acad.

Sci. USA 71,3917-3921.24. Higuchi, W. I., Prakongpan, S. & Young, F. (1973) J. Pharm. Sci.

62,945-48.25. Dam, H., Kruse, I., Prange, I., Kellehauge, H. F., Fender, H. J.

& Jensen, M. K. (1971) Z. Ernaehrungswiss. 10, 160-177.

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