THE JOURNAL cm BIOLOGICAL CHEMISTRY Vol. 245, No.20, Issue of October 25, pp. 5335-5340, 1970

Printed in U.S.A.

Inhibition of Lipolysis by Normal Alcohols

(Received for publication, May 20, 1970)

F. H. MATTSON, R. A. VOLPENHEIN, AND L. BENJAMIN

Fmn The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45239

SUMMARY

The hydrolysis of methyl oleate by pancreatic lipase (EC 3.1.1.3) is inhibited by added normal alcohols. The effi- ciency of inhibition increases with the chain length of the alcohol, attaining a maximum at 10 carbon atoms. A further increase in chain length causes no further increase in in- hibitory action.

The data have been successfully treated by assuming that the reaction occurs at an interface. The inhibitory action of the alcohols followed the pattern of a typical Langmuir adsorption isotherm. The calculated free energy of adsorp- tion of each -CH- group of the alcohol was 820 cal. Although the uninhibited reaction was enzyme-limited, the effect of the alcohols could be overcome by the addition of more substrate, but not by the addition of more enzyme. It is concluded that the inhibiting effect of alcohol is due to its adsorption on the substrate, thus blocking the enzyme from the substrate. Some of the properties of ester hydrolysis, which result from the reaction taking place at an oil-water interface, are discussed.

Numerous enzymatic reactions take place at an interface In spite of their frequent occurrence, an adequate treatment of the kinetics of such reactions has not been developed; the usual methods are not applicable to interfacial reactions because these assume that the reaction is occurring in a homogeneous solution. Although the commonly used treatments of kinetic data assume a homogenous reaction, there are at the molecular level elements of heterogenous catalysis, and indeed the Michaelis-Menten equation can be derived from the adsorption process as described by Langmuir. McLaren (1, 2) has developed some equations based mainly on the Freundlich isotherm for enzymatic reactions at a solid-liquid interface. Desnuelle’s group (3, 4) has pointed out the importance of interfacial area rather than bulk concen- tration for oil-water systems.

One such interfacial reaction, with which we have been con- cerned, is the hydrolysis of water-insoluble esters by pancreatic lipase (EC 3.1.1.3). This enzyme cannot hydrolyze water- soluble esters; apparently it requires an interface, usually oil- water, for it to be active (3). In the course of our studies, it was observed that hydrolysis is inhibited by n-alcohols.’ Examina- tion of this inhibition revealed it to be a tool by which the nature of the interfacial reaction could be at least partially delineated. For reasons that are pointed out below, the approach of Langmuir

(5) to the kinetics of interfacial reactions was selected for de- scribing the reaction.

PROCEDURE

The esters used in these studies were prepared in our labo- ratory. For the synthesis of these, the alcohols and fatty acids, except oleic acid, were obtained from commercial sources and were purified by distillation and column chromatography. The oleic acid was isolated from olive oil. The starting materials and esters were shown by gas-liquid and thin layer chroma- tography to be of greater than 99% purity. The interfacial tension of methyl oleate-aqueous 0.1 M Tris at pH 4 and 8 was 30.7 and 31.3 dynes per cm2, respectively.

The lipolytic enzymes, other than lipase, in rat pancreatic juice were inactivated by keeping a pH 9 solution of rat pan- creatic juice at 40” for 1 hour. The methods for obtaining, storing, and treating the pancreatic juice have been described previously (6).

Except where noted, the digestion mixture consisted of 225 pmoles of substrate, 330 pmoles of CaC12, 7 pmoles of free oleic acid, 0.002 M histidine, 1 M NaCl, and 1.2 mg of lyophilized rat pancreatic juice in a total volume of 55 ml at a pH of 9.0 and at 25”. All of the components except the enzyme, but including the alcohol where added, were stirred for 5 min before adding the enzyme. The rate of stirring of the digestion mixture was such that a further increase in the amount of agitation did not cause an increase in the rate of enzymatic hydrolysis (7). The gas space in the flask was continuously flushed with a stream of COz-free, water-saturated nitrogen. By carrying out the di- gestions at pH 9 all of the resulting free fatty acids are titrated (7). This pH is within the broad pH optimum of lipase (6). The course of hydrolysis of the esters was followed with the aid of a pH-stat and the use of 0.02 N KOH. The rate of KOH addition required during digestion was linear over the first several minutes. The rates of hydrolysis are expressed as microequiv- alents of free fatty acid released per min per mg of lyophilized pancreatic juice.

RESULTS

As shown in Fig. 1, the amount of methyl oleate hydrolyzed increased linearly between 0.1 and 1.2 mg of added enzyme. In Fig. 1 and in a more extensive series in Table I, it can be seen that the rate of hydrolysis did not change over a wide range of methyl oleate levels.

The percentage inhibition of the enzymatic hydrolysis of methyl oleate by various n-alcohols as a function of the amount of alcohol added to the 55 ml reaction mixture is given in Figs. 2, 3, and 4. From these graphs the amount of each alcohol

5335

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5336 Inhibition of Lipolysis by Normal Alcohols Vol. 245, No. 20

.G 4- < i2 3- 65 4 * I’ i’ ,- 0.2 0.4 0.6 0.8 1.2 1.6 20 2.4

Enzyme, mg Alcohol Added, log p moles

FIG. 1. Effect of enzyme and substrate level on methyl oleate hydrolysis. Amount of met’hyl oleate, micromoles: 0, 50; 0, 100; A, 225.

FIG. 4. Inhibition of methyl oleate hydrolysis by added decanol (A), dodecanol (C), or hexadecanol (0).

TABLE II

TABLE I

Effect of level of substrate on methyl oleafe hydrolysis

Digests contained the indicated amount of methyl oleate and 0.6 ig of enzyme.

Methyl oleate

p?nolcs

25 60

111 154 189 225 496

1000

-

_- Free fatty acid

Chain length of alcohol

peq/min/mg enzyme 3

3.3 4

3.0 5

3.3 G

3.6 7

3.6 8

3.5 10

4.0 12

3.6 16

I / I I I I I 2.8 30 3.2 34 36 3.8 4.0 4.2 44 46

Alcohol Added, log p moles

FIG. 2. Inhibition of methyl oleate hydrolysis by added pro- panol (A), butanol (O), or pentanol (0).

100

80-

l a I4 I I I I I I I I I.0 1.2 1.4 1.6 I.8 2.0 2.2 2.4 2.6 2.8 3.0

Alcohol Added, log p moles Chain Length of Alcohol

FIG. 3. Inhibition of methyl oleate hydrolysis by added hexanol FIG. 5. Amount of added alcohol causing 50% inhibition of (A), heptanol (0), or octanol (El). methyl oleate hydrolysis.

I I I I I I I I 02 0.4 06 0.8 I.0 I.2 1.4 I.6 1.8 20

Constants obtained by plotting per cent inhibition against log con- centration of alcohol (I& or by Equation 9 (K)

(micromoles per 55 ml)

I60

35,000 14,600

2,720 740 129

41.7 17.3 14.0 14.0

K

Dodecanol and various levels of methyl &ate’

3,060 780 186

33.9 14.4 10.0 13.0

Methyl &ate I60 K

pmoles

50 6.0 5.5 112 7.8 7.5 225 15.8 13.8 450 28.8 25.4

a Calculated by the two methods from the values in Figs. 2, 3 and 4.

6 Calculat,ed by the tmeo methods from the values in Fig. 6.

I , I I I I I I 2 4 6 8 IO I2 14 If

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Issue of October 25, 1970 F. H. Mattson, R. A. Volpenhein, and L. Benjamin 5337

TABLE III

Percentage inhibition of hydrolysis of various metl;

Alcohol

&lmoles Hexanol

200 500 740 960

Octanol 23 75

140 300

Decanol 5

10 20 40

Dodecanol 5

10 20 40

esters by various alcohols

Fatty acid component

Oleate Erucate Petroselenate Elaidate

%

19 38 44 51

43

26 50 84 80

17 35 42 52 64

23 29 27 36 45 53 63 67

14

57

45

80

37

57

-

41

82

30

68

41 71

TABLE IV Effect of varying level of substrate and of enzyme on hydrolysis

of methyl oleate in presence of 20 pmoles of dodecanol

Digested conditions: indicated amounts of methyl oleate and enzyme and 20 pmoles of dodecanol.

ElUtp.2 No alcohol Plus alcohol Inhibition

m&T @Eq/free fatty ocia/min % 50 pmoles of methyl oleate

0.6 1.2

1.5 3.0

1.5 3.0

0.80 1.5 3.0

0.80 1.5 3.0

0.44 0.74

71 75

100 pmoles of methyl oleate 0.6 1.2

0.55 63 1.0 67

225 pmoles of methyl oleate 0.3 0.6 1.2

0.39 0.73 1.7

51 51 44

450 pmoles of methyl oleate 0.3 0.6 1.2

0.60 25 1.1 27 2.2 27

that caused a 50% inhibition (Z& of the reaction was deter- mined. These are listed in the upper part of Table II. Values for methyl and ethyl alcohol could not be obtained, because the extremely high concentrations that were necessary grossly altered the digestion system. The log ZCO values for alcohols are plotted in Fig. 5. It will be noted that the inhibitory action of the alcohols increased with chain length up to decanol. Any further extension of the chain length of the alcohol did not increase the efficiency of inhibition.

Inhibition by the alcohols could be a function of the position of the double bond in the fatty acid. To test this possibility,

I /

Vo -Vi

/ I /

IO 20 30 1,Alcohol Added, p moles

FIG. 6. Inhibition of the hydrolysis of various amounts of methyl oleate by dodecanol. vo and vi are the rates of hydrolysis in the absence and in the presence of added alcohol. Numbers in parentheses are the slopes of the lines. Digest contained indicated amounts of methyl oleate and dodecanol and 0.6 mg of enzyme.

the effect of various alcohols when the substrate was the methyl ester of olecic acid (A9 : 10 cis), of petroselinic acid (A6:7 cis), of elaidic acid (A9: 10 truns), or of erucic acid (A13:14 cis) was determined. The values obtained are listed in Table III. The inhibition of hydrolysis was not influenced by the position or configuration of the double bond.

The effect on the rate of hydrolysis of varying the level of substrate or the level of enzyme in the presence of 20 pmoles of dodecanol was determined. The results are given in Table IV. It can be seen that the inhibitory action was not changed by the level of enzyme. On the other hand, the inhibition decreased as the amount of substrate increased. Since inhibition was related to the amount of substrate, the rates of hydrolysis of 50, 112, 225, and 450 pmoles of methyl oleate in the presence of 0, 5, 10, 15, or 30 pmoles of dodecanol were determined. The values obtained are shown in Fig. 6. The reasons for using this particular method of plotting the data are discussed below.

The inhibition of the hydrolysis of methyl oleate (225 /Imoles) by hexane, heptane, and dodecane was determined. Approxi- mately 3600 pmoles of each of these hydrocarbons were necessary to effect a 50% reduction in the rate of hydrolysis of the methyl oleate. The ZhO values for the corresponding alcohols were 740 pmoles for hexanol, 129 pmoles for heptanol, and 14 pmoles for dodecanol.

DISCUSSION

In an enzymatic reaction occurring at an interface, the sub- strate concentration is not the bulk concentration of the system, but rather the interfacial concentration. Benzonana and Desnuelle (4) have calculated the kinetics of lipolysis with the initial interfacial area of the oil phase in place of substrate concentration. Whether this area is the same when protein (lipase) has been added or whether it remains constant once the enzymatic reaction has commenced is not known. More over, in their experiments they obtained a constant interfacial area by the addition of a surface-active agent. Such a material will, of course, concentrate at and alter the interface. This was

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5338 Inhibition of Lipolysis by Normal Alcohols Vol. 245, No. 20

not acceptable in our studies in which we were trying to delineate the characteristics of reactions in an initially pure ester-water interface. Their use of surface area as the effective substrate concentration assumes that the surface contains only substrate molecules. This cannot be correct because of the presence of the surface-active agent, which will occupy a portion of the interface. The consistent results thnt they obtained indicate that the fraction of the surface occupied by substrate, although unknown, was constant among t’he various preparations. Thus, they are incorrect in expressing rates in terms of absolute surface area; rather, the areas are only relative. In our experiments too, the surface area of the substrate was not known. Thus it was not possible to determine absolute rate constants; however, several characteristics of the reaction can be described qualita- tively and relative rates can be obtained.

The system with which we were working contained an oil and a water phase. The added alcohol partitioned between the two phases and as a consequence the concentration of added alcohol in either phase was not known. To circumvent this problem, we used only a volume of 55 ml for the aqueous phase and, except where noted, 225 Fmoles of substrate, and added alcohol is reported in terms of absolute amount (micromoles), not concentration. Whereas the short chain alcohols accu- mulated mainly in the water phase, the long chain alcohols, such as dodecanol, were mainly in the oil phase, or interface; the saturation solubility of dodecanol in water is only 1.44 X 1OV mole per liter (8).

The results obtained in our experiments suggested that we were dealing with a Langmuir type of adsorption. The linear form of the solution adsorption isotherm is given by the equation

C, a -=- +c” Y Ym Ym

where C, is equilibrium bulk concentration of solute, y is amount of solute adsorbed, ym is amount of solute adsorbed in a complete monolayer, and a is kl/kz where kl and kz are proportionality constants related to desorption and adsorption. We believe that the system with which we were working was one in which

8 60- e ? 6 50-

I I IO 20 30 40

FIG. 7. Percentage inhibition of hydrolysis and per cent of sur- face covered by alcohols. The curve for fractional coverage of the surface was calculated from Equation 3 in the text with K = 14. The poir~ls are the percentage inhibition of hydrolysis by decanol (o), dodecanol (O), and hexadecanol (A) taken from Fig. 4.

the alcohol was adsorbed at the oil-water interface, the amount adsorbed being a function of the amount of alcohol present and a proport.ionality constant related to the desorption and adsorption characteristic of each alcohol. The rate of hydrolysis would be proportional to the fraction of the surface not covered by al- cohol. Substituting in the Langmuir equation yields

I K I -= v,+v, (2) vo - vi

where I is equilibrium concentration of alcohol, but as discussed in the preceding paragraph we used the absolute amount, v,, is rate of hydrolysis in the absence of alcohol, vi is rate of hydrolysis in the presence of alcohol, and K = kd/k, where kd and k, are proportionality constants related to desorption and adsorption. It follows that v0 - vi is proportional to the amount of oil surface that is covered by alcohol. A plot of I/(vO - vi) against 1 will yield a straight line, if this treatment of the experimental data is in a,ccord with the Langmuir theory. The intercept will be K/v0 and the slope 1 /va. Such a plot is used inFig. 6. Theslopes of the lines should be the same because the rate of hydrolysis of the uninhibited reaction did not change with the level of sub- strate (Table I). The conformity to theory is quite good.

Further, if our use of the Langmuir type of adsorption for the treatment of the data is correct, the following relation should hold

I

*=K+I (3)

where B is the fraction of surface covered. From this it follows that when B = 0.5, K = I. Thus the Igo values derived from Figs. 2, 3, and 4 should be the same as the K values as obtained in Equation 2, if the inhibition of the reaction is proportional to the fraction of the surface covered. Similarly, the K values obtained from the data used in Fig. 6 should be the same as the I50 value obtained for those data from a plot of the type used in Fig. 2. The 160 and K values calculated by these two methods are listed in Table II. The data for propyl and butyl alcohol did not conform to Equation 2 in that a plot of the data did not yield a straight line. The figures obtained by the two methods for the remaining alcohols and for various levels of substrate are in sufficient agreement to describe them as the same, thus confirming Langmurian adsorption. This is illustrated further by the following comparison. The fraction of the surface covered, 8, in the presence of various amounts of alcohol, i.e. a Langmuir adsorption isotherm, was calculated from Equation 3 with 14 (Table II, decanol and longer chain lengths) as a value for K. The per cent of the surface covered and the measured values for the percentage inhibition of the hydrolysis of methyl oleate by decanol, dodecanol, and hexadecanol are shown on Fig. 7. The inhibition of the enzymatic reaction by alcohol clearly has the form of a Langmuir adsorption isotherm.

The free energy of adsorption for each -CH2- group of the alcohols was calculated from the slope of the line in Fig. 5 by use of the equation

NAF, = RT In $ (4)

where AF, is the free energy of adsorption per -CH- group and C1 and Cz are the ZsO values of alcohols differing in carbon length by N -CH2- groups. The value obtained was 820

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Issue of October 25, 1970 F. H. Mattson, R. A. Volpenhein, and L. Benjamin 5339

cal. The free energy change of micellization of compounds with large polar head groups is 650 cal per -CH2-- group (9), while the values for solution (lo), desorption (II), and adsorption (12) of normal aliphatic alcohols are all about 830 cal. Thus the adsorbed alcohol in our system is at the extreme of hydrophobic bonding with no water associated with the hydrocarbon chain; that is, the hydrocarbon portion of the alcohol is roughly per- pendicular to the oil-water interface with the chain inserted in the oil (methyl oleate) phase.

There are a number of reports of hydrophobic bonding be- tween enzyme and inhibitor. However, as would be predicted (13), this interaction may be incomplete. This is reflected in the free energy changes per -CHz- group that have been reported; all are considerably less than the 820 cal observed here. Thus the free energy change per -CH2- group of the inhibitor alcohol is 650 cal for lipoxygenase (14), 400 cal for leucine amino- peptidase (15), and 560 cal for pepsin (16). Similarly, Berg- mann and Segal (17) have determined the energy change for the transfer of one -CHr group of methonium compounds from its free state in solution to adsorption on an enzyme surface to be 500 cal when the enzyme is serum cholinesterase and 300 cal when it is true cholinesterase. Main and Hastings (18) report a free energy change of 663 cal per -CH- group for the inhi- bition of serum cholinesterase by malaoxon esters.

In all of these instances, the energy changes have been at- tributed to hydrophobic bonding of the enzyme and the inhibitor. The highest value for the free energy of adsorption of one -CH*- group is 650 cal. As reported here, the value for the system in which lipase is the enzyme is 820 cal. The lower energy values found for the other enzymes indicate incomplete dehydration of the -CH2 groups when the inhibitor is ad- sorbed onto the enzyme. The phenomenon in those instances is one of inhibition of the enzyme. In our system there is com- plete dehydration of the alcohol chain which could only be at- tained if the chain was inserted into the methyl oleate drop. Thus the phenomenon that we are dealing with is blocking of the substrate.

Equation 1 strictly applies only when C, is the equilibrium concentration. Similarly, I in Equation 2 should be the equi- librium concentration of alcohol. We have expressed the added alcohol in absolute amounts rather than concentration, and the volumes of the oil and water phases were constant. As a conse- quence, in most instances, amount can replace concentration in this equation. That the values are those for the initial amount rather than at equilibrium does limit the application of this equation. Thus, the equilibrium amount is the initial amount minus the amount adsorbed at the interface. For the shorter chain alcohols relatively large quantities of alcohol were needed for inhibition and only a small portion of the total would be adsorbed at the interface. Under these conditions the equi- librium amount and the initial amount would be essentially the *ame. In the case of decanol, and the longer chain alcohols, approximately 15 pmoles of alcohol were required to bring about 50% inhibition. These alcohols are sparingly soluble in water; the solubility of dodecanol, for example, is 1 pmole/55 ml. Therefore, it seems reasonable that a major portion of t,he 15 pmoles of alcohol was adsorbed at the interface. As a conse- quence, the initial amount and the equilibrium a’mount would not be equal, and the above equation would not be applicable. The break in the curve in Fig. 5 at an alcohol having a chain length of 9 or 10 carbons is believed to be due to the use of initial

rather than equilibrium amounts; i.e. the constant I50 value for long chain alcohols is essent’ially that required to saturate half of the interface.

The results presented in Table III show that the inhibitory action of the alcohols was not influenced by the position or configuration of the double bond in the fatty acid of the sub- strate. This is in keeping with the concept that the long hydro- carbon chain of the fatty acid, anchored at the oil-water interface by the hydrophilic ester linkage, is essentially free (19). Con- sequently, the ability of an alcohol to displace equally all of these monounsaturated esters is not unexpected.

The ester used in our studies consisted of a short chain alcohol and a long chain fatty acid. The inhibitory action of hexanol and dodecanol on esters consisting of alcohols and fatty acids of short, medium, and long chain lengths was determined. Al- though there were small and apparently significant differences in inhibition, no consistent pattern was apparent. In an earlier publication (20), we presented evidence that the rate of hydrolysis of esters of various acyl and alkyl chain lengths was influenced by their orientation at the ester-water interface. If, as we are proposing here, the alcohols inhibit by acting on the substrate, then the effectiveness of that inhibition could be influenced by the orientation of the substrate at the oil-water interface. We are testing this hypothesis by other experimental techniques.

The inhibitory action of these alcohols is a function of their selective adsorption at the oil-water interface and is not simply a dilution of the substrate. The latter type of inhibitory action was seen in the case of the hydrocarbons, hexane, heptane, and dodecane. Since these are not selectively adsorbed at the inter- face, their 160 values are the same, regardless of their chain length. Moreover, the greater surface activity of the ester group of methyl oleate relative to these hydrocarbons results in the selective accumulation of the ester at the interface. As a consequence, 50% inhibition was not attained until the hydro- carbon constitutes 94 moles $& of the bulk oil phase. This is the reverse of the situation when dodecanol was the additive; there 50% inhibition was obtained when the added alcohol was equivalent to 6 moles y0 of the bulk oil phase.

The ratio of micromoles of methyl oleate to I5o, listed in the lower part of Table II, is relatively constant at approximately 15: 1. The distribution of alcohol between the bulk oil phase and the interface will be a function of the volume of the bulk phase and the area of the interface. (The water phase can be ignored because of the low water solubility of this alcohol.) That the Is,, value per mole of substrate is constant over a sub-

TABLE V Free fatty acid and hexyl oleate content after digesting methyl oleate

for various time intervals in presence of I-‘4C-hexanol

Digestion conditions as described under “Methods” except that 450 pmoles of methyl oleate and 600 pmoles of l-14C-hexanol were used.

Time Hexyi &ate Product as hexyl ester

min pntoles &H&S % 10 16 2.6 14 20 32 5.0 14 30 49 6.3 11 45 78 10.6 12 60 97 12.7 12

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5340 Inhibition of Lipolysis by Normal Alcohols Vol. 245, No. 20

strate range of 100 to 450 pmoles indicates that the volume to 160 values for C&-C& alcohols were determined with pure pan- area ratio is constant over this range, and thus the distribution creatic lipase, with a specific activity (24) of 2400 units, and of drop size did not change with these substrate levels. methyl oleate as the substrate. The 160 values obtained with

The nature of the reaction with which we are dealing makes the the pure enzyme and with the treated pancreatic juice were the data amenable to treatment by interfacial kinetics. Although same. such an approach is particularly appropriate to reactions at an interface, it is not greatly different from those generally used in enzymic studies. For example, Dixon and Webb (21) have pointed out that adsorption in accordance with the Langmuir isotherm can supply the base that will lead to the equation of Michaelis and Menten. The relationship can be seen by com- paring Equations 2 and 3 with the form of the Michaelis equation that was suggested by Hanes (22). The plot of his equation is similar to that used by us in Fig. 6.

The observed decreases in free fatty acid formation in the presence of added alcohol could be due to alcoholysis, rather than hydrolysis, taking place. Methyl oleate was digested for various periods of time in the presence of 600 pmoles of l-r*C- hexanol. The lipids were recovered from the digest and the ester fraction was isolated by column chromatography. The amount of hexyl oleate in the ester fraction was determined by assaying for radioactivity. These values and the amount of free fatty acid, determined by titration during the digestion, are shown in Table V. There was hexyl ester formation, which, regardless of the amount of methyl oleate that was lysed, amounted to about 12q/, of the digestion products. Other evidence, not reported here, led us to believe that there is com- petition between the water and the hexanol as the fatty acid acceptor. Inhibition of methyl oleate lysis and alcoholysis involves two different mechanisms. However, the occurrence of both makes complete kinetic treatment of the data impossible. This does mean that the values for free fatty acid in the other tables and figures underestimate the extent of enzymatic ac- tivity, hydrolysis plus transesterification, by approximately 12%.

Since these experiments were carried out, a satisfactory method for isolating pancreatic lipase has been reported (23). The

1.

i:

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5. 6.

7.

8.

9. 10. 11. 12.

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22. 23.

24.

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