Zinc Bioavailability from Phytate-Rich Foods and Zinc Supplements

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Zinc bioavailability from phytate rich foods and zinc supplements. Modelling the effects of food components with oxygen, nitrogen and sulfur donor ligands Ning Tang, and Leif H. Skibsted J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02998 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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Journal of Agricultural and Food Chemistry

Zinc bioavailability from phytate rich foods and zinc supplements. Modelling the effects of food components with oxygen, nitrogen and sulfur donor ligands Ning Tang and Leif H. Skibsted* Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark *Corresponding Author: Tel: 45-3533 3221; E-mail: [email protected]; ORCID: 0000-0003-1734-5016

1 ACS Paragon Plus Environment


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Abstract

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Aqueous solubility of zinc phytate (Ksp = (2.6 ± 0.2)⨯10-47 mol7/L7), essential for zinc bioavailability

3

from plant foods, was found to decrease with increasing temperature corresponding to ∆Hdis of -301 ±

4

22 kJ/mol and ∆Sdis of -1901 ± 72 J/mol K. Binding of zinc to phytate was found to be exothermic for

5

the stronger binding site and endothermic for the weaker binding site. The solubility of the slightly

6

soluble zinc citrate and insoluble zinc phytate was found to be considerably enhanced by the food

7

components with oxygen donor, nitrogen donor and sulfur donor ligands. The driving force for the

8

enhanced solubility is mainly due to the complex formation between zinc and the investigated food

9

components rather than ligand exchange and ternary complex formation as revealed by quantum

10

mechanical calculations and isothermal titration calorimetry. Histidine and citrate are promising ligands

11

for improving zinc absorption from phytate rich foods.

12

Key words:

13

titration calorimetry, density functional theory, bioavailability

Zinc phytate, zinc citrate, nitrogen donor ligands, sulfur donor ligands, isothermal

14

15

16

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18

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Introduction

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Zinc is an essential trace element involved in a variety of important biological functions such as

22

cellular metabolism, protein synthesis, wound healing and cell division.1-3 Accordingly, zinc deficiency

23

will negatively affect the physical growth, immune competence, neural development and also lead to

24

high risk of infections.4 As there are no simple markers of zinc deficiency in individuals, the exact

25

definition of zinc status remains difficult but zinc deficiency is considered as a common problem for

26

infants and children in many developing countries due to the increased zinc requirements during the

27

growth.5 According to the recent studies, dietary zinc deficiency risks are decreasing but still prevalent

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and it remains a worldwide problem affecting around 2 billion people not only restricted to the

29

developing countries but also present in the developed countries.6,7 The most common reasons of zinc

30

deficiency are inadequate dietary intake of absorbable zinc and the low bioavailability because of the

31

presence of zinc absorption inhibitor like phytic acid in plant based diets.8 The sustainable long term

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approach to improve zinc intake is dietary modification, however, this strategy involves the change of

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dietary practices and preferences which may be difficult.5,9 Moreover, more information about the zinc

34

content and bioavailability of the local foods is required to guide the people for appropriate selection of

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suitable foods. Zinc supplementation is another useful way to target vulnerable individuals at high risk

36

of zinc deficiency.10,11 Water soluble zinc salts like zinc gluconate are widely used as supplements to

37

prevent zinc deficiency due to its high solubility and bioavailability.12 Moreover, zinc gluconate is also

38

used as an ingredient for treating children diarrhea in combination with oral rehydration, common cold

39

and diseases caused by zinc deficiency.13 However, the strong metallic and bitter taste of zinc

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gluconate need to be masked when used as food supplements as the off-taste is very obvious even at

41

low dosage. In addition, the relative low zinc content of zinc gluconate results in high costs for using



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this compound.12 An alternative zinc salt with high zinc content and promising sensory properties is

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zinc citrate, which is recommend for use in syrup by WHO and has relatively low cost. Furthermore,

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the human absorption studies have demonstrated that this compound given as a supplement outside

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meals has the similar bioavailability as zinc gluconate.12

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Zinc absorption takes place in the small intestine through a not saturated carrier mediated mechanism

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under normal physiological conditions.14,15 This absorption mechanism will be affected by other factors

48

such as the chemical form of zinc salts, dietary factors and zinc status.5 Some studies have shown that

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zinc bioavailability is highly related to the specific solubility of zinc salts in aqueous solution.16,17

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Furthermore, the dietary factors like amino acids, proteins and organic acids are functioning as zinc

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absorption promoters, while phytate has been found to be the main inhibitor.8 However, the

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information about the dissolution of these zinc salts and how zinc interacts with above mentioned

53

promoters and inhibitors is very limited. In addition, the competitive binding of zinc between the food

54

components in relation to zinc absorption is not well characterized. Accordingly, the present study was

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designed to investigate the dissolution behavior of zinc salts and zinc binding mechanism of relevant

56

food components by combining isothermal titration calorimetry technique and quantum mechanical

57

calculations. In addition, the interactions between the zinc salts and food components related to zinc

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absorption were also studied. Such study hopefully will provide useful information for a better

59

understanding of zinc binding mechanism by food components, which further should aid in developing

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new zinc supplements for improving zinc bioavailability.

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Materials and methods


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Chemicals. Zinc citrate dihydrate (purity ≥ 97%), L-cysteine (purity ≥ 97%), L-cystine (purity ≥

63

99.7%), N-acetyl-L-cysteine (purity ≥ 99%), L-histidine (purity ≥ 99%), L-carnosine (purity ≥ 98%),

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L-glutathione (purity ≥ 98%), bovine serum albumin (purity ≥ 95%), D-gluconic acid sodium salt

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(purity ≥ 99%), sodium citrate dihydrate (purity ≥ 99%), phytic acid dipotassium salt of analytical

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grade, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (purity ≥ 99%), xylenol orange

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disodium salt of analytical grade and zinc standard for ion coupled plasma optical emission

68

spectrometry (in nitric acid) were all purchased form Sigma-Aldrich (Brøndby, Denmark). Zinc

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gluconate hydrate (purity ≥ 97%) was obtained from Alfa Aesar (Ward Hill, MA, USA). Zinc phytate

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was purchase from Bomei Biotechnology Company (Hefei, China). N-acetyl-L-histidine monohydrate

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(purity ≥ 99%) was obtained from TCI America Chemical Company (Tokyo, Japan). Water was

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purified by a Milli-Q Plus system (Millipore Corp., Bedford, MA, USA). All other chemicals were of

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analytical grade and were used without further purification.

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Solubility of zinc gluconate, zinc citrate and zinc phytate. Saturated aqueous solutions of zinc salts

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were prepared by adding 10 g or 25 g of zinc gluconate, 3 g of zinc citrate or 0.1 g of zinc phytate to

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100 mL of Milli-Q water, respectively, at 273 K, 286 K, 298 K, 310 K and 322 K. All samples were

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equilibrated under constant stirring using a magnetic stirrer in a thermostated water bath. The samples

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were analyzed after 120 min, 240 min, 360 min, 480 min and 1440 min of equilibration at 298 K.

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Before each analysis, the equilibrated samples were filtered (589/3, Whatman, Dassel, Germany). The

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total zinc concentration was determined by EDTA titration or inductively coupled plasma optical

81

emission spectrometry. Then solubility of each zinc salt was calculated based on the total zinc

82

concentration. All samples were prepared in duplicate.



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Solubility of zinc citrate and zinc phytate in the presence of other food components. Different

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amount of cysteine, cystine, N-acetyl-cysteine, glutathione, histidine, N-acetyl-histidine, carnosine,

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BSA, phytate and citrate powder were mixed with zinc citrate or zinc phytate, and then 100 mL Milli-Q

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water were added. These samples were equilibrated at 298 K for 120 min under constant stirring using

87

a magnetic stirrer. Before each analysis, the equilibrated samples were filtered (589/3, Whatman,

88

Dassel, Germany). Then the total zinc concentration was determined by EDTA titration or inductively

89

coupled plasma optical emission spectrometry.

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EDTA titration. The EDTA solution was first standardized by titrating EDTA solution into calcium

91

chloride solution using 0.5% murexide as an indicator. The filtered sample (1 mL or 5 mL) was

92

transferred to a titration flask and diluted by adding 20 mL of acetate buffer (pH 5.5). Then 400 uL or

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600 uL of 0.1% xylenol orange were added to the solutions as an indicator, and titrated by EDTA until

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the initial purple color changed to bright yellow indicating the end point.18 All samples were titrated in

95

duplicate.

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Ion coupled plasma optical emission spectrometry. The low zinc concentration solutions (Systems

97

contain phytate) were analyzed by ion coupled plasma optical emission spectrometry (5100, Agilent

98

Technologies, USA) coupled with a SeaSpray concentric glass nebulizer and a glass cyclonic spray

99

chamber (Limit of detection 0.22 µg/L). All filtered samples were diluted with 5% HNO3 and

100

transferred to the test tubes. The wavelength of 213.857 nm was selected for zinc determination. The

101

nebulizer and plasma flow were set at 0.7 L/min and 12 L/min, respectively. Before the injection, the

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system was rinsed using 2% mixed (HCl and HNO3) solution. The quantification of zinc concentration

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was based on the external calibrated standard curve using standard zinc solution.19 All samples were

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prepared in duplicate.


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Isothermal titration calorimetry. The isothermal titration calorimetry experiments were performed on

106

a Nano-ITC calorimeter with a gold sample cell (TA Instruments, New Castle, USA). All samples were

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degassed for 15 min before the titration using a degassing station (TA Instruments, New Castle, USA).

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The solutions in the sample cell were stirred at 250 rpm using injection syringe to ensure the rapid

109

mixing. Typically, 10 µL of titrant was injected to the sample cell over 25 s with a time interval to

110

ensure the signal return to the baseline between the injections. The control experiment was performed

111

through titrating the titrant to the buffer solution and the obtained signal was subtracted from the final

112

analysis. The data was analyzed through NanoAnalyze (TA Instruments, New Castle, USA).20

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Dynamic light scattering. The particle size was determined using a Zetasizer Nano ZSP (Malvern

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Instruments, Malvern, UK) through dynamic light scattering. The refractive index was adjusted

115

according to the measured solutions. For solutions, the samples were filtered with a 0.22 um filter and

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then 1.5 mL of filtered samples were transferred to the cuvettes with path length of 1 cm. Each sample

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was measured for 3 times and all measurements were performed at 298 K. The data was analyzed using

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Zetasizer software (Malvern Instruments, Malvern, UK) and the polydispersity index based size

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distribution is presented in the present study.21

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Quantum mechanical calculations. All quantum mechanical calculations were performed using

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Gaussian 09 package.22 Density functional theory (DFT) was employed using Becke’s three parameter

122

hybrid exchange functional along with the Lee-Yang-Parr correlation (B3LYP) in combination with

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mixed basis set due to the zinc ion. During the geometry optimization and frequency calculation, C, H,

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O, N, P and S elements were calculated using B3LYP/6-31G(d, p) basis set, while B3LYP/LanL2DZ

125

basis set was used for zinc element. The solvent effect was considered through applying the integral

126

equation formalism for the polarizable continuum (IEFPCM) model.23 The calculated frequency was



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analyzed to ensure optimized structures correspond to the local minima on the potential energy

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hypersurface.

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Results and Discussion

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Dissolution of zinc gluconate, zinc citrate and zinc phytate. The aqueous solubility of three zinc

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salts of interest for zinc bioavailability was investigated at different temperatures ranging from 273 K

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to 322 K, and the data are shown in Table 1. Among these zinc salts, zinc gluconate (Ksp = 0.058 ±

133

0.0013 mol3/L3) and zinc citrate (Ksp = (1.6 ± 0.2)⨯10-9 mol5/L5) often used as zinc supplements were

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much more soluble than zinc phytate (Ksp = (2.6 ± 0.2)⨯10-47 mol7/L7), as indicated by the apparent

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solubility product. As can be seen from Table1, the solubility of zinc gluconate was found to increase

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with increasing temperature, while the solubility of zinc citrate and zinc phytate decreased with

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increasing temperature. The overall dissolution process was accordingly endothermic for zinc

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gluconate, and was exothermic for zinc citrate and zinc phytate. The Van't Hoff equation was used to

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describe dissolution process

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LnK = −

∆

+

∆

(1)

141

based on the concentration based solubility product. According to the linear regression in agreement

142

with this equation as shown in Figure 1A, the ∆Hdis of 52 ± 1 kJ/mol, -66 ± 11 kJ/mol and -301 ± 22

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kJ/mol, ∆Sdis of 150 ± 3 J/mol K, -393 ± 40 J/mol K and -1901 ± 72 J/mol K were obtained for zinc

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gluconate, zinc citrate and zinc phytate respectively. The negative entropy of dissolution for zinc citrate

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and zinc phytate indicates the ordering effect of water molecules by these zinc salts exceeds the

146

disorder induced by the dissolution of their crystals due to the highly charged citrate and phytate ions

147

as such ordering effects depend on the magnitude of the charge on each ion. The thermodynamics of


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the results are in agreement with previously obtained results for calcium salts, which the dissolution

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process for calcium gluconate and calcium citrate was found to be endothermic and exothermic,

150

respectively.24,25 In addition, the least soluble zinc phytate of three investigated zinc salts exhibited the

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highest temperature sensitivity with the solubility ratio of 21 for a temperature interval of 322 K.

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Similar results were also found for calcium salts as the less soluble salts showed higher temperature

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sensitivity.24 The extremely low solubility of zinc phytate indicates an precipitation when dietary zinc

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interacts with phytate from plant based diet in the gut leading to a low zinc bioavailability, which is in

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agreement with previous studies demonstrating that phytate as one of the main inhibitors for zinc

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absorption.8 Moreover, the large particle size of zinc phytate (around 207 ± 20 nm) as determined by

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dynamic light scattering also indicated the poor solubility and bioavailability.26

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Binding of zinc to gluconate, citrate and phytate as investigated by isothermal titration

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calorimetry and density functional theory calculations. The thermodynamic parameters for

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formation of zinc gluconate, citrate and phytate complexes were investigated using an isothermal

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titration calorimetry technique combined with density functional theory calculations, and the data are

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presented in Table 2, Table 3, Figure 2 and Figure 3. Gluconate (Table 2 and Figure 2) was found to

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form the weakest zinc complex with exothermic binding process as ∆Hass < 0 obtained through

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independent binding model (red line in Figure 2D), which is in accordance with previously reported

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weak zinc binding ability by gluconate using differential pulse polarography.27 Zinc binding ability of

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gluconate decreases with increasing temperature, while the solubility of zinc gluconate increases. These

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two opposite effects together strongly increase the zinc ion concentration in aqueous solution with

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increasing temperature. The driving force for formation of zinc citrate was much stronger with a

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binding constant of (1.1 ± 0.2)⨯104 L/mol at pH 7.4 and 298 K, which is in good agreement with a



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previously obtained value of (3.47 ± 0.13)⨯104 L/mol at pH 6 and 298 K using the same technique.28

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This entropy driven binding process (∆Hass = 5.4 ± 0.3 kJ/mol, ∆Sass = 95.1 J/mol K) balanced partly

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the decreasing solubility of zinc citrate as should be reflected in a less decrease in zinc activity. Similar

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temperature effects have been found for calcium citrate.25 In contrast to the endothermic association

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process for zinc citrate, binding of zinc to phytate was found to be more complicated with multiple

175

binding sites as determined by isothermal titration calorimetry as shown in Figure 2 C and F. The

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integrated heat flow data was processed by multiple sites binding model, and values for the

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corresponding thermodynamic parameters are presented in Table 2. For the stronger binding site, the

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binding process was exothermic with a binding constant of (1.4 ± 4)⨯106 L/mol at pH 7.4 and 298 K,

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but the driving force did not decrease with increasing temperature. Such a surprising association

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behavior may relate to the effect of the weaker endothermic zinc binding site as shown in Table 2

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(Phytate Site 2) and Figure 2 (C and F). Furthermore, the change in heat capacity, ∆CP, defined as

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∆C = ∆

(2)

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could be characteristic for conformational changes by metal ion binding to a large molecule such as

184

phytate. In addition, it was calculate to characterize the temperature dependence of the enthalpy of zinc

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binding by linear regression of equation (2) as shown in Figure 1B. Such temperature dependence of

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enthalpy has been shown to associate with surface desolvation upon binding, and to a lesser extent,

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with the difference in vibrational modes between the complex and the free species.29,30 The positive

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value of ∆CP for zinc gluconate and zinc citrate obtained from the slope of Figure 1B may indicates

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that the dehydration of gluconate ions, citrate ions and zinc ions is order forming meaning that the

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complex formation is entropy driven and decreases with increasing temperature as seen for citrate. In


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contrast, the negative value for zinc phytate (both binding sites) confirms the hydration of the highly

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negative charged phytate group. The structures of 1:1 binding complexes were calculated by density

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functional theory using B3LYP/6-31G(d, p) and LanL2DZ mixed basis set combined with polarizable

194

continuum model. The optimized zinc complexes in aqueous solution are shown in Figure 3, from

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which it can be seen that two oxygen atoms, one from carboxylate group and one from hydroxyl group

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(C10), were involved in zinc binding for forming zinc gluconate. In contrast, zinc coordinated to citrate

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and phytate with three oxygen atoms with the bond length around 2.0 Å to form tridentate structures

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(Table 3 and Figure 3). The shortest zinc-oxygen bond length of 1.94 Å, as shown in Table 3, was

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found in zinc/phytate complex corresponding to the strongest zinc binding affinity. Similar results were

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also seen in calcium binding as shorter length of calcium-oxygen bonds corresponding to stronger

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calcium binding.23 Due to the large error in estimation of Gibbs free energy in the calculations, the

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more accurate enthalpy of binding (∆Hbinding) was calculated in the present study which reported to be

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more reliable according to previous studies. As can be seen from Table 3, the ordering of zinc binding

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affinity based on the calculated binding enthalpy is in good agreement with the experimental data (ITC)

205

which demonstrated that phytate exhibited highest zinc binding ability with ∆Hbinding of -425.91 kJ/mol,

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followed by citrate (∆Hbinding of -362.14 kJ/mol) and gluconate (∆Hbinding of -106.89 kJ/mol). In

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addition, as shown in Table 3, the relative driving force for zinc binding of citrate and phytate was 3.3

208

and 4, respectively, obtained from DFT calculations, which is also in fair agreement with

209

experimentally determined value of 2.4 and 3.6 confirming the validity of the optimized structures.

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Zinc citrate and zinc phytate solubility enhancement through interacting with food components

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with oxygen, nitrogen and sulfur donor ligands. Dietary factors like amino acids, peptides, proteins

212

and other low molecular weight ions are known to form soluble complex with zinc enhancing zinc



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bioavailability as the solubility of zinc is increased in the digestive tract. These ligands have been

214

widely used for improving zinc absorption and the knowledge regarding these dietary factors has

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expanded substantially in recent years. Further knowledge is, however needed regarding the

216

interactions between the dietary factors in relation to the zinc absorption from meals and mixed diets.

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The slightly soluble zinc citrate and insoluble zinc phytate were selected to investigate the interactions

218

with other food components that are related to zinc absorption in order to understand the competitive

219

binding of zinc between these food components for better zinc bioavailability. Thermodynamic

220

parameters for zinc binding by these food components are shown in Table S1. As can be seen from

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Table S1, these food components all exhibited relatively strong zinc binding affinity with exothermic

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binding. For sulfur donor ligands (cysteine, cystine, N-acetyl-cysteine and glutathione), thiol groups

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and carboxylate groups were involved in zinc binding as indicated by the optimized complex structures

224

showed in Figure S1, while for nitrogen donor ligands (histidine, N-acetyl-histidine and carnosine),

225

imidazole group and carboxylate group were involved. Notably, there is a discrepancy between the

226

calculated zinc binding affinity and experimental data as indicated by ∆Hbinding and ∆Grel (Table S1).

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This difference may relate to the pH effect due to the pH was adjusted to 7.4 for all isothermal titration

228

calorimetry experiments. In addition, among the investigated food components, the zinc transporter

229

protein bovine serum albumin (BSA) exhibited the strongest zinc binding ability with the binding

230

constant of 2.3⨯105 L/mol, followed by cysteine (6.5⨯104 L/mol) and cystine (3.1⨯104 L/mol).20 The

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interactions between these food components and zinc citrate in relation to the competitive binding of

232

zinc were investigated in more details through the dissolution of zinc citrate in the presence of different

233

amount of the investigated food components in aqueous solution and the results are shown in Table 4

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and Figure 4. As revealed by solubility product presented in Table 4, stable zinc citrate solutions with

235

enhanced solubility were found to be formed in all cases except for cystine within the equilibrium time 12 ACS Paragon Plus Environment

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(2 h) by dissolution of excess zinc citrate in already saturated zinc citrate solutions at 298 K. In contrast,

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a decrease of solubility of zinc citrate was observed when adding solid cystine during the dissolution

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(Table 4 and Figure 4B), this may be due to the extremely low aqueous solubility of cystine. Although

239

cystine has strong zinc binding ability (Table S1), the driving force of dissolved cystine for zinc

240

binding was not strong enough to change the equilibrium of zinc citrate due to the extremely low

241

aqueous solubility of cystine. For the other sulfur donor ligands (cysteine, N-acetyl-cysteine and

242

glutathione), the formed zinc citrate solutions with enhanced solubility were very stable and no

243

precipitation was observed during the storage up to 3 months. As show in Table 4, among these ligands,

244

N-acetyl-cysteine was the most efficient ligand for improving the solubility of zinc citrate by a factor of

245

3.05 when 0.0092 mol solid N-acetyl-cysteine were added during the dissolution. Moreover, the pH of

246

the zinc citrate solution with enhanced solubility decreased with increasing amount of sulfur donor

247

ligands. This pH decrease may facilitate the dissolution of the excess zinc citrate as the pH was found

248

to drop more dramatically for N-acetyl-cysteine. Under the actual experimental conditions for sulfur

249

donor ligands, the maximal degree of solubility enhancement of 3.23 corresponding to a solubility of

250

1.27 ± 0.03 g/100 mL was observed when 0.0825 mol solid cysteine were added. In addition, the

251

increase in zinc citrate solubility was significant as seen by comparing the solubility product presented

252

in Table 4. The solubility increase was quantified by the degree of solubility enhancement (czn2+/cZn02+)

253

defined as the zinc concentration ratio between the final solutions and saturated solutions. The degree

254

was found to depend linearly on the amount of added sulfur donor ligands (Figure 4) under the

255

investigated conditions. Similar linearity was also found for dissolution of excess calcium lactate by

256

citrate which was explained by a mechanism entailing an initial binding of citrate to the calcium lactate

257

surface.31 Accordingly, the dissolution of excess zinc citrate by sulfur donor ligands was assumed to be

258

related to a similar mechanism appearing as a zero order reaction with a constant rate in proportion to 13 ACS Paragon Plus Environment


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the amount of added ligands. Compared with sulfur donor ligands, the nitrogen donor ligand, histidine,

260

was found even more efficient for increasing the solubility of zinc citrate without affecting the pH of

261

the dissolution. As shown in the insert picture in Figure 4E, the presence of 0.0645 mol solid histidine

262

resulted in dissolution of 3 grams of solid zinc citrate in 100 mL of water corresponding to the degree

263

of solubility enhancement of 7.13 (Table 4). For the other nitrogen donor ligands (N-acetyl-histidine

264

and carnosine), enhanced zinc citrate dissolution was also demonstrated with different degrees of

265

solubility enhancement depending on the amount of added ligands. Notably, there was a decrease in

266

solubility of zinc citrate when 0.0021mol carnosine was added during the dissolution (Table 4 and

267

Figure 4G). This may due to the pH increase as the final solution pH increased to 7.24, as shown in

268

Table 4. In addition, in contrast to the results for sulfur donor ligands, the pH of nitrogen donor ligands

269

induced final solutions, in general was found to increase with increasing amount of added ligands.

270

Furthermore, as can be seen from Figure 4, the degree of solubility enhancement was also found to

271

depend linearly on the amount of added ligands. Based on a previous dissolution study for calcium

272

citrate,25 the dissolution of zinc citrate was assumed to follow a similar stepwise reaction with three

273

species accordingly dominating in the saturated solutions:

274

Zn3Cit2.2H2O → Zn2+ + 2ZnCit- +2H2O (3)

275

ZnCit- ⇌ Zn2+ + Cit3- (4)

276

The presence of sulfur donor or nitrogen donor ligands (L-) with high zinc binding ability will lower

277

the free zinc concentration due to the complex formation (Table S1):

278

Zn2+ + L- ⇌ ZnL+

(5)


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In addition, ternary complex may also formed during the equilibrium as citrate has a comparable zinc

280

binding ability:

281

ZnCit- + L- ⇌ ZnCitL2-

(6)

282

ZnL+ + Cit3- ⇌ ZnLCit2- (7)

283

According to above equilibrium, the strong zinc binding ability of the nitrogen and sulfur donor ligands

284

and an apparent high solubility of the formed complexes (ZnL+) together ensure the robust

285

enhancement of solubility of the zinc salts. For the protein ligand, BSA, more complicated effect was

286

observed. As shown in Table 4 and Figure 4 H, the solubility of zinc citrate was found to decrease in

287

the presence of less than 7.58 ⨯ 10-5 mol of solid BSA during the dissolution. This solubility decrease

288

may relate to the BSA nanoparticle formation (sphere shape) due to the interaction between citrate and

289

BSA blocking the binding site of BSA for zinc binding and a reduced solvation of zinc citrate. The

290

formation of BSA nanoparticles was demonstrated by dynamic light scattering as may be seen from

291

Figure 6. Moreover, the obtained BSA nanoparticles exhibited different size distribution (Table 4 and

292

Figure 6) depending on the molar ratio between zinc citrate and BSA. Previous studies have reported

293

the successful use of BSA nanoparticle for delivery carriers, but the size of the used BSA nanoparticle

294

was not prepared in a controlled manner.32 Above results indicated that the size of formed BSA

295

nanoparticles may be controlled by changing the molar ratio between zinc citrate and BSA. The size

296

controlled BSA nanoparticles could be of importance for delivery system, but it needs to be studied in

297

more details. Notably, as shown in Table 4 and Figure 6, when 1.52⨯10-4 mol (10g) BSA was used

298

during the dissolution, no BSA nanoparticles were found to be formed but enhanced zinc citrate

299

solubility was observed as indicated by the solubility product. This is an example of not forming BSA



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nanoparticles because of changing the molar ratio and the dissolved large amount of BSA exhibited

301

strong driving force for changing the equilibrium (3) and (4) due to zinc binding resulted in further

302

dissolution of zinc citrate. In addition, the effect of phytic acid on solubility of zinc citrate was also

303

investigated. As can be from Table 4, the presence of phytic acid was also found to assist the

304

dissolution of zinc citrate but it should be noted that the formed zinc complex may not absorbable and

305

accordingly this effect may not be important for improvement of zinc absorption.

306

Compared with zinc citrate, the solubility enhancement induced by the investigated food components

307

was much more remarkable for insoluble zinc phytate with the degree of solubility enhancement more

308

than 17000 (Table 5). For sulfur donor ligands, cystine was also found to inhibit the dissolution of zinc

309

phytate which is in agreement with the results for zinc citrate. Among the other sulfur donor ligands,

310

N-acetyl-cysteine showed the highest ability for assisting the dissolution of zinc phytate resulted in 0.1

311

g of zinc phytate completely dissolved in water corresponding to the highest degree of solubility

312

enhancement, as can be seen from Table 5 and Figure 5C. This observed high efficiency for dissolving

313

excess zinc phytate by N-acetyl-cysteine may be partially attributed to the resulted low pH of the

314

formed final solution (Table 5) as zinc phytate has higher solubility at low pH. As presented in Table 5,

315

cysteine and glutathione also efficiently enhanced the dissolution of excess zinc phytate with the degree

316

of solubility enhancement around 12000. In addition, the pH of the final solution was also found to

317

decrease with increasing amount of cysteine and glutathione. Moreover, as presented in Figure 5, the

318

degree of solubility enhancement seems to depend linearly on the amount of added sulfur donor ligands

319

under the investigated conditions which is in accordance with previous results for zinc citrate indicating

320

the a similar reaction mechanism. For nitrogen donor ligands, in contrast to the results for zinc citrate

321

and sulfur donor ligands, the maximal degree of solubility enhancement was observed when histidine


Page 17 of 55


322

was added during the dissolution of zinc phytate. According to the non-linear regression showed in

323

Figure 5E (red line), the maximal degree of solubility enhancement of 12226 ± 6389 was obtained.

324

Moreover, the zinc phytate solutions with enhanced solubility were also found to be formed when

325

adding the other nitrogen donor ligands (N-acetyl-histidine and carnosine) during the dissolution but

326

less efficient than the sulfur donor ligands as indicated by the degree of solubility enhancement (Table

327

5). This is partly related to the solution pH increase as the pH of the final zinc phytate solutions with

328

added nitrogen donor ligands were much higher than the pH of the final solutions with sulfur donor

329

ligands but close to the physiological pH which is more important for zinc absorption, as shown in

330

Table 5. As can be seen from Table 5 and Figure 5H, a fluctuation in solubility is noted when adding

331

BSA during the dissolution of zinc phytate. This fluctuation may still due to the BSA nanoparticle

332

formation which may be seen from the size distribution (Table 5), as highly negatively charged phytate

333

ions similar to citrate ions indicating similar nanoparticle formation mechanism. However, the

334

observed nanoparticle formation was not as evident as for the citrate ion induced nanoparticle

335

formation because of the low solubility of zinc phytate. In addition, citrate, reported as a zinc

336

absorption promoter, was also found to dissolve excess zinc phytate in already saturated zinc phytate

337

solutions. As shown in Table 5, the solubility enhancement in zinc phytate was very significant as

338

indicated by the solubility product. Similar to histidine, the maximal degree of solubility enhancement

339

was also observed for citrate, as can be seen from Figure 5I. Notably, the pH of the final zinc phytate

340

solutions was close to 7.4 despite of the low pH range of citrate solutions providing further information

341

for citrate as a zinc absorption enhancer.

342

Reaction mechanism for the solubility enhancement as investigated by isothermal titration

343

calorimetry and density functional theory calculations. As zinc phytate is not soluble, the slightly



Page 18 of 55

344

soluble zinc citrate was selected for competitive binding study in order to understand the above

345

observed solubility enhancement better. Isothermal titration calorimetry was used to monitor the

346

reactions by titrating zinc citrate solution with cysteine (sulfur donor ligand), histidine (nitrogen donor

347

ligand) and BSA (protein). Table 6 shows the obtained thermodynamic parameters, compared with the

348

reactions by titrating zinc chloride with same ligands, lower binding constant values were observed for

349

cysteine and histidine which is in agreement with competitive binding of zinc between citrate and

350

ligands leading to the binding of zinc to cysteine and histidine weaker. In addition, the decrease in

351

entropy was also observed for both enthalpy driven reactions. As shown in Table 6 and Figure S2, BSA

352

exhibited two different binding patterns. The exothermic reaction corresponds to the binding of zinc to

353

BSA, while the endothermic reaction corresponds to the binding of citrate to BSA providing further

354

information of BSA nanoparticle formation due the interaction between citrate and BSA. A multiple

355

binding sites model was applied to fit the obtained data and a value of (3.0 ± 2.2)⨯107 L/mol was

356

obtained for binding of citrate to BSA indicating a strong binding. According to the solubility

357

enhancement results, the nitrogen donor ligand, histidine, exhibited strong ability for dissolving excess

358

zinc citrate or zinc phytate in already saturated solutions leading to the enhanced solubility with the

359

resulted solution pH around physiological pH which is of importance for zinc absorption in gut.

360

Accordingly, the reaction of binding of zinc to histidine in the presence of different amount of citrate

361

ions or phytate ions were also investigated using isothermal titration calorimetry. The results are shown

362

in Table 6 and Figure 7, the presence of citrate ions or phytate ions did not cause significant changes in

363

the heat flow as can be seen from Figure 7. Even in the system of equal molar concentration of citrate

364

or phytate and histidine, the obtained heat flow is still similar to the results of titrating histidine with

365

zinc chloride. Moreover, when citrate or phytate is dominant in the system, it seems that the zinc still

366

reacts with histidine initially which is more evident for citrate (Figure 7C). Based on the previous 18 ACS Paragon Plus Environment

Page 19 of 55


367

results, binding of zinc to histidine is an exothermic process while to citrate is an endothermic process,

368

and both reactions are following a 1:1 binding pattern. Therefore, the zinc binding constant for

369

histidine in the presence of citrate can be quantified by using multiple sites binding model. As shown in

370

Figure 7 (D, E and F), the multiple binding sites model was successfully applied to the obtained data,

371

from which the binding constants were obtained and presented in Table 6. Compared with the binding

372

constant obtained from the system without citrate ions (Table S1), the presence of citrate ions greatly

373

improved the zinc binding ability of histidine with the average binding constant of 3.66⨯104 L/mol

374

which is significantly higher than 6.4⨯103 L/mol (without presence of citrate ions). In addition, as

375

shown in Table 6, zinc binding by citrate became much weaker with the average binding constant of

376

954 L/mol which is in agreement with the observed heat flow demonstrated that zinc reacted with

377

histidine first due to the much stronger binding affinity in the presence of citrate ions. Although the

378

binding constants can not be quantified for phytate containing system, it seems that the reactions were

379

still following the similar pattern which may be seen from Figure 7 (G, H and I). Notably, the above

380

obtained apparent thermodynamic parameters are much dependent on the experimental conditions and

381

sometimes may not comparable. Therefore, another method based on an overall equilibrium in the

382

system was applied to investigate the binding constant further. In the present study, zinc is known to

383

form 1:1 complex with histidine and citrate, and histidine is mainly presented in three forms (A2-, HA-

384

and H2A) in the solution at pH 7.4 by considering the pKa value of carboxylate group and imidazole

385

group. Accordingly, the overall equilibrium with ignoring the interaction between zinc and Tris-HCl

386

buffer may be described by the following equation:

387 388

(1-αZnCit-)Zn2+ + αZnCit-ZnCit- + (1- αHA- - α )A2- + αHA-HA- + αH2AH2A ⇌ ZnA +( αHA- + 2α )HCit2- + (αZnCit- - αHA- -2α )Cit3- (8) 19 ACS Paragon Plus Environment


Page 20 of 55

389

where α is the molar fraction of different species in the overall equilibrium. Therefore, two kinds of

390

competitive binding reactions involving zinc binding and proton binding exist in the overall

391

equilibrium. The following equations were also applied to calculate the zinc binding affinity:

392

Czn2+ = [Zn2+] + [ZnCit-] + [ZnA]

393

CA = [A2-] + [HA-] + [H2A] + [ZnA] K

394

!"#

= [&][

[ !"# ]

!"'# ]

K # = [ &][#]

396

K = [ &][ (13) #]

397

(10)

(11)

395

[ # ]

(9)

(12)

[ ]

The apparent zinc binding constant was calculated using the following equation K = [&]

[]

398

()* [

# ]

()*

(14)

399

Where [Zn2+]ITC and [A2-]ITC are the concentrations of zinc species and histidine species not forming

400

complex [ZnA], respectively. Accordingly, their concentrations can be calculated using the following

401

equations derived from equation (9)-(14):

402

[Zn2+]ITC = [Zn2+] + [ZnCit-] = [Zn2+](1+KZnCit-[Cit3-]) (15)

403

[A2-]ITC = [A2-] + [HA-] + [H2A] = [A2-] + KHA- [H+][A2-] + K KHA- [H+]2[A2-] (16)

404

According to the above equations, then the binding constant of 9.3⨯104 L/mol can be obtained from the

405

following equation:


Page 21 of 55


406

KFinal = K(1+KZnCit-[Cit3-])(1+ KHA- [H+] + K KHA- [H+]2) (17)

407

Compared with the binding constant of 1.1⨯104 L/mol (citrate) obtained from ITC (Table 2), histidine

408

still exhibited stronger zinc binding ability. Both methods indicated that histidine had stronger zinc

409

binding ability than citrate when they were presented together in the solution. Such difference in the

410

binding constant may lead to a complete zinc chelation by only histidine which is in agreement with the

411

observed heat flow showed in Figure 7. In addition, the other possible driving forces such as ligand

412

exchange and ternary complex formation for forming solutions with enhanced solubility were also

413

investigated through quantum mechanical calculation. The ternary complex structures were optimized

414

using B3LYP/6-31G(d,p) and LanL2DZ mixed basis set combined with the solvent effects and the

415

results are shown in Figure S3 and S4. As can be seen from Figure S3 and S4, for zinc citrate, the

416

ternary complex formation with cysteine and N-acetyl-cysteine was accompanied by proton transfer

417

from thiol group to carboxylate group which is in agreement with the decreased pH in the final

418

solutions. Notably, the ternary complex between zinc phytate and cysteine can not be calculated as the

419

formed complex was not stable due to the interaction between phytate and cysteine which was found to

420

be very slow as the equilibrium can not be established as monitored by isothermal titration calorimetry.

421

In addition, the ternary complex zinc citrate/histidine was not formed according to the calculated

422

formation enthalpy, as shown in Table 7. According to the calculated enthalpy for both driving forces

423

presented in Table 7, the enthalpy for ligand exchange were all positive indicating the reaction would

424

not happen in the solution, and accordingly was not the driving force for the solubility enhancement.

425

This result also confirms that the zinc binding ability by citrate and phytate are stronger than the

426

investigated food components. However, negative enthalpy for ternary complex formation was

427

obtained suggesting the complexes exist in the final solutions. Compared with the ∆Hbinding for forming



Page 22 of 55

428

ZnL+ (Table S1), the absolute ∆H values showed in Table 7 were much smaller indicating ternary

429

complex formation was not the main driving force for solubility enhancement and the main species in

430

the solution were ZnL+, which is very promising for zinc absorption as the ternary complex containing

431

phytate may not be absorbed by the human body. Accordingly, it seems very clear that the main driving

432

force for forming the solutions with enhanced solubility was the complex formation for ZnL+ due to the

433

much higher zinc binding ability of the investigated food components in the presence of citrate ions or

434

phytate ions. Metal binding ability of the ligands is related to their chemical hardness or softness as

435

indicated by the Pearson's hard soft acid base theory which is the hard-hard and soft-soft combinations

436

are thermodynamically favoured over crossed interactions.33 Accordingly the related parameters were

437

also calculated using the same mixed basis set for a better understanding of zinc binding by the

438

investigated food components. Following Koopmans' theorem, the above mentioned parameters were

439

calculated according to the following equations:34,35

440

441

χ = − μ = η =

-.

-7 /

/

=

= −

01232 .04532 /

04532 701232 /

(18)

(19)

442

where χ is the electronegativity indicating the ability of attracting electrons; µ is the chemical potential;

443

I is the ionization potential; A is the electron affinity;

444

electrophilicity index (ω) and chemical softness (σ) were calculated based on the following equations:36

445

ω = /: (20)

446

σ = /: (21)

η is the chemical hardness. Then, the

9
= / ⨯

Zinc Bioavailability from Phytate-Rich Foods and Zinc Supplements

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