Jacobs Journal of Veterinary Science and Research

Effect of Benzoic Acid and its Chemical Analogues on Membrane Resistance against Osmotic Pressure in Isolated Cattle Erythrocytes: A Comparative Study with Other Animal Species

*Hitoshi MINEO
Faculty Of Human Science, Hokkaido Bunkyo University, Japan

*Corresponding Author:
Hitoshi MINEO
Faculty Of Human Science, Hokkaido Bunkyo University, Japan
Email:mineo@do-bunkyodai.ac.jp

Published on: 2019-04-01

Abstract

We determined the effects of benzoic acid and its derivatives on osmotic fragility (OF) in cattle red blood cells (RBCs) in vitro. Cattle RBCs were exposed to these substances at 0-100 mM in a buffer solution for 1 hour and then the 50% hemolysis was determined by soaking in 0.2-0.8% NaCl solution. OF was determined as the NaCl concentration inducing 50% hemolysis, for which was colorimetrically measured by concentration of released hemoglobin. Benzoic acid, heptanoic acid and cyclohexanecarboxylic acid tended to decrease OF, but this change was not statistically significant (N.S). Replacement of COOH bound to the benzene ring with PO(OH)2 or SO2OH abolished the OF response obtained by benzoic acid. Replacement with OH did not affect the OF response up to 25mM, but induced hemolysis at 50 and 100 mM. Replacement with CONH2 decreased OF and the degree of the OF-lowering effect was larger than that of benzoic acid. Some derivatives substituted with other groups (OH, CH3 or NH3) or halogens (Cl or Br) decreased or tended to decreased OF, with the degree of change in OF dependent on the positon of the substitution on the benzene ring. 2-, 3- and 4-Hydroxybenzoic acids tended to decrease OF, but these changes were N.S. 2- and 4-Aminobenzoic acids, but not 3-aminobenzoic acid, decreased OF dose-dependently. 3- and 4-Methylbenzoic acids, but not 2-metylbenzoic acid, significantly decreased OF dose-dependently. 2-, 3- and 4-Chrolobenzoic acids, and 2-, 3- and 4-bromobenzoic acids significantly decreased OF dose-dependently. The effects of the substitution with other elements on OF were dependent on the elements and their position on the benzene ring. Regression analysis using the value of all compounds tested revealed that there were significant negative correlations between the partition coefficient of compounds and their effects on OF response at 25, 50 and 100 mM. Although OF responses to benzoic acid and its derivatives in cattle RBCs were similar to those in guinea pig and sheep RBCs, they differed markedly from those in rat RBCs.

Keywords

Benzoic Acid; Partition Coefficient; Osmotic Fragility; Erythrocyte; Membrane; Fatty Acid; Arachidonic Acid; Cattle

Introduction

Benzoic acid is an aromatic chemical and a simple carboxylic acid composed of a carboxylic group and a benzene nucleus. In terms of physicochemical characteristics, benzoic acid is an amphiphilic substance possessing a hydrophilic carboxylic group and a hydrophobic benzene nucleus. The most well-known action of benzoic acid is its anti-microbial effect, and it is used as a preservative in various processed foods. Benzoic acid itself is basic material from which many derivatives have been chemically produced for various purposes. Salicylic acid, one of the chemical analogues of benzoic acid, is generally used as an anti-febrile and painkilling drug. Carboxylic esters of benzoic acid and its analogues are widely used as artificial fragrances in cosmetics and perfumes.

The first target in physiological or pharmacological action of benzoic acid and its analogues is thought to be the cell membrane. Since mammalian red blood cells (RBCs) possess a fundamental cell membrane structure but no nucleus or intracellular organelle, they have been used as an experimental prototype, particularly of the phospholipid bilayer of the cell membrane. There are many reports that osmotic fragility (OF), as evaluated by the degree of hemolysis in RBCs, is a beneficial tool for evaluating the actions of many substances on the cell membrane in vitro. It was demonstrated that general [5] and local anesthetics [6], certain kinds of drugs [7] and toxins [8], and inorganic [9] and organic compounds [10] induce changes in OF in the RBCs of mammalian species. It has been reported that the application of salicylic acid and its derivatives induce morphological change in human RBCs [11] and these substances directly permeate artificial and human RBC membrane and interact with the phospholipid bilayers

In our previous studies, we have demonstrated that benzoic acid and most of its analogues decrease membrane resistance to osmotic pressure and decrease OF in rat RBCs [13]. In contrast, benzoic acid and some of analogues increase membrane resistance to osmotic pressure and decrease OF in guinea pig [14] and sheep RBCs [15] In terms of the response to monocarboxylic acids possessing straight, branched or cyclic hydrocarbons, OF was increased in rat RBCs [16, 17], but was unchanged or rather decreased in guinea pig [17], sheep [18] and cattle RBCs [19]. We suspected that arachidonic acid expressed as 20:4 (5, 8, 11, 14) was one of the candidates responsible for differences in OF response to monocarboxylic acids, including benzoic acid, among the RBCs from different animal species [16, 17]. As benzoic acid is also a monocarboxylic acid, it was speculated that the difference in OF response to benzoic acid and its derivatives demonstrated in the RBCs from different animal species is similarly due to differences in the composition of the RBC membrane among animal species. 

Benzoic acid and its derivatives are assumed to enter the cell membrane and interact with the phospholipid matrix, leading to changes in OF in RBCs. The infiltration of these compounds into the cell membrane is considered to be the most important factor inducing the subsequent sequence of events leading to changes in OF. The partition coefficient is one of physicochemical values showing the delivery of hydrophilic/hydrophobic balance of chemicals and is described as the ratio of the concentrations of a substance between two solvents [20]. As n-octanol is nearer in nature to the biological membrane, which is mainly composed of phospholipids, than other non-solvent solvents [21], the octanol/water partition coefficient has been commonly utilized for an indicator of the distribution of chemicals into cells, tissues and the whole body [22-24]. The Log P values evaluated as the logarithm of the partition ratio of chemicals between octanol and water are available for various chemical substances on a number of websites [25-27]

The objective of this series of experiments was to clarify the effects of benzoic acid and its analogues on OF in cattle RBCs and compare these OF responses among animal species examined in our laboratory to date. In addition, as the composition of fatty acids in cattle RBCs was already reported, we sought to clarify whether the OF response in cattle RBCs could be predicted or not based on the data for arachidonic acid content. In this study, we also clarified whether there is a relationship between the partition coefficients of benzoic acid and its derivatives and the degree of changes in OF in cattle RBCs induced by application of these chemicals. The results of these experiments allow us to con-sider the interaction of various chemicals and phospholipids in the cell membrane composed of various acyl-chains derived from fatty acids

Materials and Methods

Reagents

Biochemical grade benzoic acid and its derivatives were similar to those in our previous report [15] and purchased from Tokyo Kasei Kogyo Co., Ltd (Tokyo, Japan) or Wako Pure Chemical Co., Ltd. (Osaka, Japan). The chemical structures of these substances are shown in the tables in the Results section. All other reagents used in this study were of analytical grade

Preparation of Cattle RBCs

Cattle used in the present experiments were similar to those in our previous report [19]. Holstein female cattle aged 7 to 8 years old (n=6, body weight 400-450 kg) and fed in the Student-training Farm of Rakuno Gakuen University (Ebetsu, Hokkaido) were used for the experiments. Animals were allowed free access to concentrated feed and hay as well as tap water. Blood samples from the animals were collected by a veterinarian from Rakuno Gakuen University. The sampling and treatment of blood specimens were performed as follows. Blood samples (30 ml) were drawn by venipuncture from the left jugular vein into a heparinized test tube. The blood samples were carried to the laboratory of Hokkaido Bunkyo University and then kept in a refrigerator at 4º C for about 18 hours. Plasma and buffy coat were removed by centrifugation at 2000 g for 15 min (Model 2420, Kubota Inc., Tokyo, Japan) followed by aspiration of the upper layer. The crude RBCs thus obtained were then washed three times with two volumes of cold 0.9% NaCl solution. The resultant packed RBC suspension was kept in ice-cold water until subsequent use

Experimental Procedure

The experimental procedures followed those described in our previous report [19]. Briefly, the packed RBC suspension (30 μl) was transferred into 0.6 ml of phosphate-NaCl buffer solution (pH7.4) containing each of the carboxylic acids at 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 o100 mM in 1.5-ml micro test tubes (Nichiryo Co., Ltd., Tokyo, Japan). A suitable amount of NaCl was added to the buffer solution to adjust the osmolality for each substance tested. The RBC suspensions treated with the carboxylic acids were incubated by shaking (1 stroke/sec) at 37º C for 1 hr (Shaking Bath TBK 202 DA, Advantec Co., Ltd., Tokyo, Japan). Each RBC suspension was gently mixed using a mixer (Vortex Genie 2, Model-G560, Scientific Industry, Inc. NY., USA) following incubation, and 50 μl of each suspension was transferred into a 96-deep-well microplate (2 ml volume, Whatman Inc., Piscataway, NJ, USA) containing 1 ml of NaCl solution ranging from 0.1 to 0.8%. The deep-well microplate was immediately centrifuged at 1300 g (Plate Spin II, Kubota Inc., Tokyo, Japan) for 10 min at room temperature. The supernatants (200 μl) containing different concentrations of hemoglobin derived from the ruptured RBCs were transferred into another 96-well microplate (300 μl volume, Whatman Inc., Piscataway, NJ, USA) and determined colorimetrically at 540 nm (Microplate Reader Model 680, Bio-Rad Laboratories, Tokyo, Japan)

Statistical Analysis 

As complete hemolysis and no hemolysis of the RBC suspensions are induced in 0.1% and 0.8% NaCl solutions, respectively, the hemoglobin concentration in the 0.1% and 0.8% NaCl solutions were defined as 100 and 0%. The concentration of the NaCl solution inducing 50% hemolysis (EC50) of the treated RBCs was calculated from the hemolysis curve by using a straight-line equation between the points immediately adjacent to 50%. OF in the RBCs was defined as the EC50 value. All values are expressed as means ± S.D. (n=6). The significance of the differences between the control (0 mM) and subsequent concentrations (0.1- 100mM) was calculated by Dunnett’s test following one-way ANOVA. As apparent changes in OF were found by treatment with most of carboxylic acids at 10, 25, 50 and 100 mM, the differences from the control value at 0 mM were calculated and expressed as ΔEC50 (NaCl %). The partition coefficients of the carboxylic acids examined were mainly quoted from the chemical and physical properties on the PubChem [25], ChemSpinder [26] or ChemIDplus [27] websites. The relationship between the partition coefficient of each carboxylic acid and the ΔEC50 of the RBCs was confirmed by regression analysis. Statistical analyses were performed using Excel Tokei for Windows 2012 (SSRI Co., Ltd., Tokyo, Japan). Statistical significance was fixed at a P value < 0.05 or 0.01

Figure 1: Effects of benzoic acid and its derivatives on OF in cattle RBCs. The effects of (upper panel): benzoic acid (?), heptanoic acid (?), and cyclohexanecarboxyolic acid (?), (middle panel) benzenephosphonic acid (?) and benzenesulphonic acid (?), and (lower panel) benzamide (?) and phenol (?) are presented. Values are the means ± SD (n=6). Open symbols indicate that there was a significant difference between the control (0 mM) and subsequent concentrations (0.1-100 mM) based on Dunnett’s test (P<0.05, including the case of P

  

Results

Substitution of the Benzene Ring or Carboxylic Group by other elements

Figure 1 A shows changes in OF in cattle RBCs by application of benzoic acid (C6 H5 -COOH) and its derivatives in which the benzene ring (C6 H5 ) was replaced by a straight hydrocarbon chain (C6 H13) or cyclohexane ring (C6 H11). Although benzoic acid tended to decrease OF in a dose-dependent manner, this change was not statistically significant (N.S). Heptanoic acid (C6 H13-COOH) and cyclohexane carboxylic acid (C6 H11-COOH), both of which have 6 carbons, also tended to decrease OF, but these changes were N.S. Benzenephosphonic acid (C6 H5 -PO(OH)2 ) and benzenesulfonic acid (C6 H5 -SO2 OH) had no effect on OF at any dose (Figure 1B). Benzamide (C6 H5 -CONH2 ) decreased OF in a dose-dependent manner from 25 to 100 mM (Figure 1C). Hydroxybenzene (C6 H5 -OH) had no effect on OF from 0.1 to 25 mM, but induced hemolysis at 50 and 100 mM (Figure 1C). The partition coefficients of the substances tested and their effects on OF expressed as EC50 values at 10, 25, 50 and 100 mM are summarized in Table 1.

Substitution of Hydrogen on the Benzene Ring by Other Groups 

The effects of the replacement of hydrogen (H) by a hydroxyl (OH), amino (NH2 ) or methyl (CH3 ) group at the 2, 3 or 4 position on the benzoic ring are shown in Figure 2. Application of 2-,3- or 4-hydroxybenbenzen tended to decrease OF in cattle RBCs (Figure 2A), but these changes were N.S. 2- and 4-Aminobenzoic acids, but not 3-amonobenzoic acid, decreased OF in a dose-dependent manner with statistical significance (P<0.05) (Figure 2B). Although 2-methylbenzoic acid had no effect on OF, 3- and 4-methylbenzoic acids significantly decreased OF in a dose-dependent manner (P<0.05) (Figure 2C). Partition coefficients and the effects of substances on OF are shown in Table 2.

Substitution of Hydrogen on the Benzene Ring by a Halogen

The effects of the replacement of hydrogen (H) on the benzene ring by chlorine (Cl) or bromine (Br) atom at the 2-, 3- or 4-position on OF in cattle RBCs are shown in Figure 3. 2-, 3- or 4-Chrolobenzoic acid decreased OF dose-dependently (P<0.05) (Figure 3A). 3-Bromobenzoic  

   Table :1 Effects of benzoic acid and its derivatives in which replaced the benzene ring or carboxylic group was replaced with another element on OF in cattele RBcs 

Values are means ± SD (n=6). The partition coefficients were obtained from the PubChem [25], ChemSpider [26] or ChemIDplus [27] website. The ΔEC50 values (degree of change in NaCl %) at 10, 25, 50 and 100 mM are presented. Asterisks (* and **) indicate that there was a significant difference  (P<0.05 and P <0.01) between the control (0 mM) and
subsequent concentration (0.1-100 mM) based on the Dunnett’s test.

acid decreased OF dose-dependently (P<0.05). Although OF was decreased dose-dependently by 2- or 4-bromobenzoic acid up to 50 mM (P<0.05), the OF value was slightly increased at 100 mM. Partition coefficients and the effects of substances on OF are shown in Table 2.

Relationship between the Partition Coefficient of the Substances and their effect on OF

Table 4 shows the partition coefficients of the tested substances and their effects on OF (ΔEC50) by treatment at 10, 25 50 and 100 mM. To clarify the relationship between the two factors, regression analyses were performed using each table at 10, 25, 50 and 100 mM. Significant negative correlations (P<0.05) were demonstrated between the partition coefficient and ΔEC50 at 25, 50 and 100 mM in cattle RBCs.

Discussion

In this series of experiments, we clarified the effects of the application of benzoic acid and its derivatives at 0.1 to 100 mM for one hour on OF in cattle RBCs. Application of benzoic acid tended to decrease OF in cattle RBCs, but this change was N.S. Replacement of the benzene ring with a straight hydrocarbon chain or cyclohexane ring had no effect on the OF response induced by benzoic acid. With regard to the replacement of the carboxylic group by other elements, benzenephosphonic acid tended to increase OF

Table 2: Effects of benzoic acid derivatives in which H was replaced with a OH, NH3 or CH3 on the benzene ring on OF in cattle RBCs.

Values are means ± SD (n=6). The partition coefficients were obtained from the PubChem [25], ChemSpider [26] or ChemIDplus [27] website. The ΔEC50 values (degree of change in NaCl %) at 10, 25, 50 and 100 mM are presented. Asterisks (* and **) indicate that there was a significant difference (P

from 10-50 mM, but these changes were not N.S. Benzenesulphonic acid had no effect on OF at 0.1-100 mM. Phenol (hydroxybenzene) did not change OF from 0.1 to 25 mM, and then abruptly induced hemolysis at 50 and 100 mM. Benzamide decreased OF dose-dependently, with a significant decrease in OF observed at 100 mM. With regard to the replacement of the hydrogen on the benzene ring by another element, 2-, 3- and 4-hydoroxybenzene induced almost the same OF response as that by benzoic acid (N.S.).

Figure:2.Effects of benzoic acid derivatives in which H was replaced at 3 positions on the benzene ring with OH (upper panel), NH2 (middle panel) or CH3 (lower panel) on OF in cattle RBCs. Values are the means ± SD (n=6). Open symbols indicate that there was a significant difference between the control (0mM) and subsequent concentrations (0.1-100mM) based on Dunnett’s test (P<0.05, including the case of P

Although 2- and 3-aminobenzoic acids decreased OF in a dose-dependent manner (P<0.05), 4-aminobenzoic acid tended to decrease OF, although this change was N.S. 3- and 4-Methylbenzoic acids also decreased OF dose-dependently (P<0.05), while 2-aminobenzoic acid tended to decrease OF, although this change was N.S. 2-, 3- and 4-Chrolobenzoic acids decreased OF dose-dependently (P<0.05), and 2-, 3- and 4-bromobenzoic acid also decreased OF dose-dependently below 50 mM (P<0.05), but slightly increased the OF value in a rebounded manner at 100 mM. The results of regression analysis demonstrated that significant negative relationships exist between the partition coefficients of benzoic acid and its derivatives, and their effects on OF at 25, 50 and 100 mM (P<0.05). 

A comparison with the previous results using rat, guinea pig and sheep RBCs revealed that the OF responses in cattle RBCs to benzoic acid and its derivatives were much closer to those in guinea pig and sheep RBCs [16, 17], but differed markedly from those in rat RBCs [15]. In the experiment using rat RBCs, benzoic acid and most of its derivatives increased OF in a dose-dependent manner, with the degree of increase in OF dependent on the derivative [15]. Among the derivatives used in the present study, benzenephosphonic acid, benzamide, 2- and 3-aminobenzoic acids, and 2- and 3-hydoroxybenzoic acids did not have any effect on OF in rat RBCs [15]. Although phenol had no effect from 0.1-25 mM and induced hemolysis at 50 and100 mM in cattle RBCs, the same effects were observed in rat and guinea pig RBCs [15, 16].

In our series of experiment, we noticed that the OF response to monocarboxylic acids differed among the RBCs from different animal species, and we were able to categorize the responses into 2 types. Base on the two animal species in which we first found different OF responses in RBCs to monocarboxylic acids, we named two different types of OF responses the “rat type” and “guinea pig type” [18, 19]. In rat-type RBCs, most monocarboxylic acids increased OF in a dose-dependent manner, such rat-type OF response have been only confirmed in rat RBCs to date [18, 19]. In contrast, in guinea pig-type RBCs, monocarboxylic acids had no effect on OF or rather decreased OF, and such OF responses have been demonstrated in guinea pig [17], sheep [18] and cattle RBCs [21]. Benzoic acid and its derivatives tested in this experiment are included in the mono-carboxylic acid group, and the OF responses to these compounds were also characterized according to the rat-type and guinea pig-type responses mentioned above [19]. The present study showed that the OF responses in cattle RBCs to benzoic acid and its derivatives can also be categorized as guinea pig type.

As to the cause of differences in OF response to monocarboxylic acids, we assumed that the differences in the composition of the acyl-chains derived from fatty acids are an important factor. We predicted that arachidonic acid is a potential contributor to the 2 different types of OF response to monocarboxylic acids in RBCs.

Figure 3: Effects of benzoic acid derivatives in which H was replaced at 3 positions on the benzene ring with Cl (upper panel) or Br (lower panel) in cattle RBCs. Values are the means ± SD (n=6). Open symbols indicate that there was a significant difference between the control (0 mM) and subsequent concentrations (0.1-100 mM) based on Dunnett’s test (P<0.05, including the case of P

The first important point is the proportion of arachidonic acid in the fatty acids in the RBC membranes. The proportion of arachidonic acid is the highest among the various fatty acids contained in the phospholipid layer of the RBCs in rats [28, 29]. In addition, the proportion of arachidonic acid in rat RBCs is much higher than those in guinea pig, sheep and cattle RBCs [28, 29]. The large amounts and high proportion of arachidonic acid could be related to the specific structure of the RBC membrane, leading to the specific functional characteristics in rat RBCs described below. The second important point is the specific molecular structure of arachidonic acid as a polyunsaturated fatty acid expressed as 20:4 (5, 8, 11,14). Arachidonic acid possesses 20 carbons and four cis-double bonds in its hydrocarbon chain. Thus, it presents a particularly crooked acyl-chain structure in its moiety. This sharp bend is expected to perturb the rigid bindings of the acyl-chains aligned linearly in the phospholipid layer of the RBC membrane.

It is reported that long-chain cis-unsaturated fatty acids and their alcohol analogues, including arachidonyl alcohol, increase the membrane fluidity of cattle platelets [30]. This same report also clarified that compared to the corresponding unsaturated fatty acid, the tested saturated fatty acids and trans-unsaturated fatty acid had much lower or no effects on membrane fluidity. It was also shown that the positions of the double bonds had less influence than did the number of double bonds [30]. It was reported that the membrane fluidity is an important factor influencing Rb+ (K+ ) efflux in RBCs isolated from seven mammalian species (cows, horses, pigs, humans, rabbits, cats and rats) [31]. The rate constant of Rb+ (K+ ) efflux could be correlated with the order parameter obtained from electron spin resonance as well as with the number of double bonds in the fatty acids in membrane phospholipids. Among the six animal species, excluding humans, rat RBCs showed the highest rate constant of Rb+ (K+ ) efflux and number of double bonds in the fatty acids in the RBC membrane [31]. Further study advanced these observations and examined the relationship between the rate constant of Rb+ (K+ ) efflux and the percentage of arachidonic acid in the RBC membranes of six mammalian species (cows, horses, pigs, humans, rabbits and rats) [32]. As a result, rat RBCs demonstrated the highest Rb+ (K+ ) efflux and percentage of arachidonic acid

Table 3: Effects of benzoic acid derivatives in which H was replaced hydrogen with a Cl or Br on the benzene ring on OF in cattle RBCs.

Values are means ± SD (n=6). The partition coefficients were obtained from the PubChem [25], ChemSpider [26] or ChemIDplus [27] website. The ΔEC50 values (degree of change in NaCl %) at 10, 25, 50 and 100 mM are presented. Asterisks (* and **) indicate that there was a significant difference (P

In our series of experiment, we noticed that the OF response to monocarboxylic acids differed among the RBCs from different animal species, and we were able to categorize the responses into 2 types. Base on the two animal species in which we first found different OF responses in RBCs to monocarboxylic acids, we named two different types of OF responses the “rat type” and “guinea pig type” [18, 19]. In rat-type RBCs, most monocarboxylic acids increased OF in a dose-dependent manner, such rat-type OF response have been only confirmed in rat RBCs to date [18, 19]. In contrast, in guinea pig-type RBCs, monocarboxylic acids had no effect on OF or rather decreased OF, and such OF responses have been demonstrated in guinea pig [17], sheep [18] and cattle RBCs [19]. Benzoic acid and its derivatives tested in this experiment are included in the monocarboxylic acid group, and the OF responses to these compounds were also characterized according to the rat-type and guinea pig-type responses mentioned above [19]. The present study showed that the OF responses in cattle RBCs to benzoic acid and its derivatives can also be categorized as guinea pig type.

As to the cause of differences in OF response to monocarboxylic acids, we assumed that the differences in the composition of the acyl-chains derived from fatty acids are an important factor. We predicted that arachidonic acid is a potential contributor to the 2 different types of OF response to monocarboxylic acids in RBCs.

The first important point is the proportion of arachidonic acid in the fatty acids in the RBC membranes. The proportion of arachidonic acid is the highest among the various fatty acids contained in the phospholipid layer of the RBCs in rats [28, 29]. In addition, the proportion of arachidonic acid in rat RBCs is much higher than those in guinea pig, sheep and cattle RBCs [28, 29]. The large amounts and high proportion of arachidonic acid could be related to the specific structure of the RBC membrane, leading to the specific functional characteristics in rat RBCs described below. The second important point is the specific molecular structure of arachidonic acid as a polyunsaturated fatty acid expressed as 20:4 (5, 8, 11,14). Arachidonic acid possesses 20 carbons and four cis-double bonds in its hydrocarbon chain. Thus, it presents a particularly crooked acyl-chain structurein its moiety. This sharp bend is expected to perturb the rigid bindings of the acyl-chains aligned linearly in the phospholipid layer of the RBC membrane. 

It is reported that long-chain cis-unsaturated fatty acids and their alcohol analogues, including arachidonyl alcohol, increase the membrane fluidity of cattle platelets [30]. This same report also clarified that compared to the corresponding unsaturated fatty acid, the tested saturated fatty acids and trans-unsaturated fatty acid had much lower or no effects on membrane fluidity. It was also shown that the positions of the double bonds had less influence than did the number of double bonds [30]. It was reported that the membrane fluidity is an important factor influencing Rb+ (K+ ) efflux in RBCs isolated from seven mammalian species (cows, horses, pigs, humans, rabbits, cats and rats) [31]. The rate constant of Rb+ (K+ ) efflux could be correlated with the order parameter obtained from electron spin resonance as well as with the number of double bonds in the fatty acids in membrane phospholipids. Among the six animal species, excluding humans, rat RBCs showed the highest rate constant of Rb+ (K+ ) efflux and number of double bonds in the fatty acids in the RBC membrane [31]. Further study advanced these observations and examined the relationship between the rate constant of Rb+ (K+ ) efflux and the percentage of arachidonic acid in the RBC membranes of six mammalian species (cows, horses, pigs, humans, rabbits and rats) [32]. As a result, rat RBCs demonstrated the highest Rb+ (K+ ) efflux and percentage of arachidonic acid among the RBC membranes of the tested mammalian species, again excluding humans. The same report demonstrated that no significant correlation was observed between the rate constants and the relative contents of the four major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyeline). Another report showed that human RBCs show more K+ leakage if the fatty acid bound to the native phosphatidylcholine is partly replaced by arachidonic acid [33]. The results of those experiments suggest that a large amount of arachidonic acid may induce an increase in membrane fluidity and K+ leakage from the RBC membrane. Taken together, the results of those reports suggest that that a large amount of arachidonic acid inhibits the formation of rigid between acyl-chains and expands the space in the phospholipid matrix in the RBC membrane.

The partition coefficient is a physicochemical characteristic that is used as an indicator of the permeation of chemicals into the cell membrane [20]. Regression analysis revealed a significant negative relationship between the partition coefficients of the tested substances and the degree of change in OF at 25, 50 and 100 mM in cattle RBCs. In our previous experiment on the effect of benzoic acid and its derivatives on OF in sheep RBCs, a significant negative relationship was observed between the partition coefficients and the OF response within a limited range of concentrations [15]. For monocarboxylic acids, including straight, branched and cyclic hydrocarbons, however, no definite relationship between the above two parameters was demonstrated in guinea pig [34] and sheep RBCs [18]. On the other hand, in rat RBCs, a significant positive relationship was observed between the two parameters for monocarboxylic acids with straight and cyclic hydrocarbon chains [35]. These results indicated that, depending on the type of chemical as well as the concentration applied, the partition coefficient can be used to explain the effects of monocarboxylic acids on OF responses in RBCs. We think, however, that it is difficult to evaluate comprehensively the OF response to the whole range of monocarboxylic acids based only on their partition coefficients. This difficulty is thought to be due to the differences in the nature of the RBC membrane based on differences in the fatty acid composition.

Although the octanol/water partition coefficient has been commonly used for explaining the delivery of various chemicals into cell, tissues or the whole body [22-24], the action of many chemical compounds on a biological or artificial membrane composed of phospholipids does not necessarily follow their octanol/water partition coefficients [36-38]. This phenomenon can be explained by the fact that the conditions of the membrane, including the type of the head group or the fatty acids composition in the phospholipids, or the cholesterol content in the RBC membrane interacting with the applied compounds [39-42]. Thus, the differences in the composition of fatty acids in the RBC membrane among animal species would also have an effect in the OF response induced by monocarboxylic acids.

The benzoic acid and its derivatives used in the present study have a hydrophilic carboxylic group and a hydrophobic hydrocarbon in their moiety. Compounds possessing amphiphilic characteristics are generally thought to be a detergent. Biphasic effects on the cell membrane have been reported for various kinds of detergents; i.e. a stabilizing effect at low and a hemolytic effect at high concentrations [43-46]. The OF-lowering effect demonstrated by benzoic acid and some of its derivatives may be a membrane stabilizing effect as observed for detergents applied at low concentrations as described above. In this experiment using 2- or 4-bromocarboxylic acid, OF was decreased dose-dependently from 0.1 to 50 mM and then slightly increased in a rebound manner in cattle RBCs. This rebound OF response to some benzoic acid derivatives applied at high concentrations was also demonstrated in guinea pig [14] and sheep RBCs [15].

With regard to the protective effect of amphiphilic compounds against hemolysis, two explanations have been presented to date [47]. It has been reported that various amphiphilic compounds, including various type of detergents or anesthetics, increased the cell volume or membrane area in the RBCs [48, 49]. The swelling of RBCs is thought to be one reason for protective effect against hemolysis. On the other hand, it has been reported that the anti-hemolytic effect of amphiphilic compounds against osmolality occurs without swelling of the RBCs [50, 51]. It is thought that the permeation of compounds into the RBC membrane changes the ion balance between inside and outside of the cell membrane and inhibits hypotonic hemolysis [52, 53]. It is also reported that the cell lysis induced by the swelling in cells or changes in ion balance depends on the type of compound applied [54].

As the permeation of amphiphilic compounds into the cell membrane affects various physiological cell behaviors, such as endocytosis [55], membrane protein movement [56], or cell aggregation [57], benzoic acid and its derivatives are also speculated to possess the potential to affect functions in various cell types. The relationships between the phospholipid composition and the derivatives of benzoic acid, and the subsequent responses in the RBC membrane have to be clarified in detail. Further experiments using RBCs from various animals, apart from rats, guinea pigs, sheep and cattle are needed to clarify the effect of benzoic acid and its derivatives on osmotic resistance in RBCs. 

References

  1. Brul S, Coote P. Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int J Food Microbiol 1999; 50(1-2): 1-17.
  2.  Clissold SP. Aspirin and related derivatives of salicylic acid. Drugs 1986; 32( 4): 8-26.
  3.  Rastogi SC, Schouten A, de Kruijf N, et al. Contents of methyl-, ethyl-, propyl-, butyl- and benzylparaben in cosmetic products. Contact Dermatitis 1995; 32(1): 28- 30.
  4.  Johnson W, Bergfeld WF, Belsito DV, et al. Safety assessment of benzyl alcohol, benzoic acid and its salts, and benzyl benzoate. Int J Toxicol 2017; 36 (3): 5S-30S
  5. Toker K, Ozer NK, Yalçin AS, et al. Effect of chronic halothane exposure on lipid peroxidation, osmotic fragility and morphology of rat erythrocytes. J Appl Toxicol 1990; 10(6): 407-409.
  6. Bazzoni G, Rasia M. Effect of tetracaine chlorhydrate on the mechanical properties of the erythrocyte membrane. Blood Cells Mol Dis 2001; 27(2): 391-398
  7. Cruz Silva MM, Madeira VM, Almeida LM, et al. Hydroxytamoxifen interaction with human erythrocyte membrane and induction of permeabilization and subsequent hemolysis. Toxicol In Vitro 2001; 15(6): 615-622.
  8. Cusinato DA, Souza AM, Vasconcelos F, et al. Assessment of biochemical and hematological parameters in rats injected with Tityus serrulatus scorpion venom. Toxicon 2010; 56(8): 1477-1486.
  9. Abad C, Carrasco MJ, Piñero S, et al. Effect of magnesium sulfate on the osmotic fragility and lipid peroxidation of intact red blood cells from pregnant women with severe preeclampsia. Hypertens Pregnancy 2010; 29(1): 38-53.
  10. Quan GB, Han Y, Liu MX, et al. Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose. Cryobiology 2011; 62(2): 135-144.
  11. Li A, Seipelt H, Müller C, et al. Effects of salicylic acid derivatives on red blood cell membranes. Pharmacol Toxicol 1999; 85(5): 206-211.
  12. Suwalsky M, Belmar J, Villena F, et al. Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes. Arch Biochem Biophys 2013; 539(1): 9-19.
  13. Mineo H, Ogita A, Kanayama N, et al. Effect of the chemical specificity of benzoic acid and its analogs on osmotic fragility in erythrocytes of Sprague-Dawley rats in vitro. Eur J Pharmacol 2013; 702(1-3): 142-148
  14. Mineo H, Kasai K, Makihara R, et al. Effects of benzoic acid and its analogues on osmotic fragility in guinea pig erythrocytes in vitro. J Membr Sci Technol 2016; 6(4): 161.
  15. Mineo H, Matsuda C, Suzuki Y, et al. Benzoic acid and its derivatives increase membrane resistance to osmotic pressure in isolated sheep erythrocytes. Biochem Pharmacol (Los Angel) 2018; 7(4): 260.
  16.  Mineo H, Hara H. Chemical specificity in short-chain fatty acids and their analogues in increasing osmotic fragility in rat erythrocytes in vitro. Biochim Biophys Acta 2007; 1768(6): 1448-1453.
  17.   6(3): 156- Mineo H, Kasai K, Makihara R, et al. Monocarboxylic Acids and dicarboxylic acids induce different responses in terms of osmotic fragility in rat and guinea pig erythrocytes in vitro. J Membra Sci Technol 201163.
  18. Mineo H, Noji M, Watanabe Y, et al. Carboxylic acids with certain molecular structures strengthen the cell membrane against osmotic pressure in sheep erythrocytes in vitro. Biochem Pharmacol (Los Angel) 2018; 7(3): 252
  19. Mineo H, Moriyoshi M. Carboxylic acids with certain molecular structures decrease osmotic fragility against osmotic pressure in cattle erythrocytes in vitro: Appearance of a wedge-like effect similar to RBCs in other animal species. Biochem Pharmacol (Los Angel) 2019; 8(1): 264.
  20. Missner A, Pohl P. 110 years of the Meyer-Overton rule:predicting membrane permeability of gases and other small compounds. Chemphyschem 2009; 10(9-10): 1405-1414.
  21.  Flynn GL. Structural approach to partitioning: Estimation of steroid partition coefficients based upon molecular constitution. J Pharm Sci 1971; 60(3): 345-353.
  22. Eugene Kellogg G, Abraham DJ. Hydrophobicity: is LogP(o/w) more than the sum of its parts?. Eur J Med Chem 2000; 35(7-8): 651-661.
  23.  Mazák K, Noszál B. Drug delivery: a process governed by species-specific lipophilicities. Eur J Pharm Sci 2014; 62: 96-104.
  24.  Buchwald P, Bodor N. Octanol-water partition: searching for predictive models. Curr Med Chem 1998; 5(5): 353-380
  25.  PubChem (2018) Accessed December 14, 2018.
  26.  ChemSpinder (2018) Accessed December 14, 2018.
  27.  ChemIDplus (2018) Accessed December 14, 2018.
  28. Wessels JM, Veerkamp JH. Some aspects of the osmotic lysis of erythrocytes. 3. Comparison of glycerol permeability and lipid composition of red blood cell membranes from eight mammalian species. Biochim Biophys Acta 1973; 291(1): 190-196.
  29. Horrobin DF, Huang YS, Cunnane SC, et al. Essential fatty acids in plasma, red blood cells and liver phospholipids in common laboratory animals as compared to humans. Lipids 1984; 19(10): 806-811.
  30. Kitagawa S, Endo J, Kametani F. Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change. Biochim Biophys Acta 1985; 818(3): 391-397.
  31. Erdmann A, Bernhardt I, Herrmann A, et al. Species-dependent differences in the influence of ionic strength on potassium transport of erythrocytes. The role of membrane fluidity and Ca2+ . Gen Physiol Biophys 1990; 9(6): 577-588.
  32. Bernhardt I, Seidler G, Ihrig I, et al. Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition. Gen Physiol Biophys 1992; 11(3): 287-299
  33. Kuypers FA, Roelofsen B, Op den Kamp JA, et al. The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine. Biochim Biophys Acta 1984; 769(2): 337-347..
  34. Mineo H. Effects of carboxylic acids on osmotic resistance in rat and guinea pig red blood cells in vitro: The relationship between partition coefficient and changes in osmotic fragility. Biochem Pharmacol (Los Angel) 2018; 7(2): 245.
  35. Mineo H. Relationship between partition coefficients of carboxylic acids and changes in osmotic resistance in rat erythrocytes in vitro. Biochem Pharmacol (Los Angel) 2017; 6(3): 236.
  36.  Palm K, Stenberg P, Luthman K, et al. Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharm Res 1997; 14(5): 568-571.
  37. Balon K, Riebesehl BU, Müller BW. Drug liposome partitioning as a tool for the prediction of human passive intestinal absorption. Pharm Res 1999; 16(6): 882-888.
  38. Zhu C, Jiang L, Chen TM, et al. A comparative study of artificial membrane permeability assay for high throughput profiling of drug absorption potential. Eur J Med Chem 2002; 37(5): 399-407
  39.  Ahyayauch H, Larijani B, Alonso A, et al. Detergent solubilization of phosphatidylcholine bilayers in the fluid state: influence of the acyl chain structure. Biochim Biophys Acta 2006; 1758(2): 190-196.
  40. Zhang J, Hadlock T, Gent A, et al. Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization. Biophys 2007; J 92(11): 3988-4001.
  41. Nazari M, Kur1. Nazari M, Kurdi M, Heerklotz H. Classifying surfactants with respect to their effect on lipid membrane order. Biophys 2012; J 102(3): 498-506.
  42. Okamura E, Takechi Y, Aki K. Uptake of sevoflurane limited by the presence of cholesterol in the lipid bilayer membrane: a multinuclear nuclear magnetic resonance study. J Oleo Sci 2014; 63(11): 1149-1157.
  43. Trägner D, Csordas A. Biphasic interaction of Triton detergents with the erythrocyte membrane. Biochem J 1987; 244(3): 605-609.
  44. . Vives MA, Macián M, Seguer J, et al. Hemolytic action of anionic surfactants of the diacyl lysine type. Comp Biochem Physiol C 1997; 118(1): 71-74.
  45. Galembeck E, Alonso A, Meirelles NC. Effects of polyoxyethylene chain length on erythrocyte hemolysis induced by poly[oxyethylene (n) nonylphenol] non-ionic surfactants. Chem Biol Interact 1998; 113(2): 91-103.
  46.  Martínez V, Sánchez L, Busquets MA, et al. Disturbance of erythrocyte lipid bilayer by amino acid-based surfactants. Amino Acids 2007; 33(3): 459-462.
  47. Manaargadoo-Catin M, Ali-Cherif A, Pougnas JL, et al. Hemolysis by surfactants - A review. Adv Colloid Interface Sci 2016; 228: 1-16.
  48. Isomaa B, Hägerstrand H, Paatero G, et al. Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes. Biochim Biophys Acta 1986; 860(3): 510-524.
  49.  Mac hleidt H, Roth S, Seeman P. The hydrophobic expansion of erythrocyte membranes by the phenol anesthetics. Biochim Biophys Acta 1972; 255(1): 178-189.
  50. Beresford RA, Fastier FN. Effects of some S-alkylthiouroniums and related compounds on the osmotic fragility and the membrane expansion of human erythrocytes. Br J Pharmacol 1980; 71(1):
  51. Eskelinen S, Saukko P. The hypotonic hemolysis and the protective action of lysophosphatidylcholine. Biorheology 1984; 21(3): 363-377.
  52.  Miseta A, Bogner P, Szarka A, et al. Effect of non-lytic concentrations of Brij series detergents on the metabolism-independent ion permeability properties of human erythrocytes. Biophys J 1995; 69(6): 2563-2568
  53. . 53. Preté PS, Gomes K, Malheiros SV, et al. Solubilization of human erythrocyte membranes by non-ionic surfactants of the polyoxyethylene alkyl ethers series. Biophys Chem 2002; 97(1): 45-54.
  54. Sánchez L, Martínez V, Infante MR, et al. Hemolysis and antihemolysis induced by amino acid-based surfactants. Toxicol Lett 2007; 169(2): 177-184.
  55.  Fogler WE, Gersten DM, Fidler IJ. Inhibition by tertiary amine local anesthetics of phagocytosis in cultured mouse peritoneal macrophages. Biochem Pharmacol 1978; 27(20): 2447-2453.
  56.  Klausner RD, Bhalla DK, Dragsten P, et al. Model for capping derived from inhibition of surface receptor capping by free fatty acids. Proc Natl Acad Sci USA 1980; 77(1): 437-441.
  57. Kanaho Y, Kometani M, Sato T, et al. Mechanism of inhibitory effect of some amphiphilic drugs on platelet aggregation induced by collagen, thrombin or arachidonic acid. Thromb Res 1983; 31(6): 817-831.