
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00785-5
10.1016/j.psj.2024.104206
104206
METABOLISM AND NUTRITION
In vitro bioaccessibility of inorganic and organic copper in different diets
Wu Min *†1
Tan Guofeng †1
Shi Ruirui ‡
Chen Dewen *
Qin Yumei †
Han Jianzhong hanjz99@zjgsu.edu.cn
†2
⁎ Institute of Ecology & Health , Hangzhou Vocational & Technical College, Hangzhou 310018, China
† School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China
‡ Institute of Chemical Product Inspection, Zhejiang Fangyuan Test Group Co., LTD, Hangzhou 310013, China
2 Corresponding author: hanjz99@zjgsu.edu.cn
1 These authors equally contributed to this article.

10 8 2024
11 2024
10 8 2024
103 11 10420614 5 2024
7 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In poultry diets, copper is an essential nutrient that is critical for various physiological functions. Although copper sulfate is commonly used due to its cost-effectiveness, organic copper sources are gaining popularity because of their superior production outcomes and environmental benefits. Nevertheless, understanding the distinct bioaccessibility of inorganic and organic copper in diverse dietary setting remains limited. This study investigated the bioaccessibility of copper sulfate, copper amino acid chelate, and copper proteinate in the intestine via in vitro digestion and in situ dialysis. The results showed significant differences in the molecular size distribution of compounds formed by different copper salts within the intestinal environment, thereby leading to varying bioaccessibility. Copper sulfate has a bioaccessibility of 47 % ± 4%, which is significantly lower than copper amino acid chelate and copper proteinate (63% ± 5%, and 60% ± 4%, respectively) in purified diet systems. Similarly, in whey protein systems, sulfate records 54% ± 10% bioaccessibility compared to 78% ± 9% and 76% ± 5% for copper amino acid chelate and copper proteinate. Coexisting feed ingredients have a significant impact on copper bioaccessibility. Copper sulfate forms precipitates, reducing its bioaccessibility to 34% ± 1% in sodium nitrate solution. The addition of digestive enzyme increases the bioaccessibility of copper sulfate to 81% ± 2% by providing organic ligands. Digestive enzyme also enhanced the bioaccessibility of copper proteinate from 36% ± 4% to 81% ± 4% by degrading its ligands. However, feed ingredients may decrease copper bioaccessibility by forming macromolecular complexes with copper, as all the organic ligands can competitively bind with copper in the intestine. These findings emphasize the importance of considering copper salt types and diet composition in animal nutrition practices.

Key words

Bioaccessibility
in vitro digestion
organic and inorganic copper salt
dietary composition
poultry nutrition
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pmcINTRODUCTION

Copper plays a particularly significant role in poultry diets, crucial for maintaining health and meeting essential nutritional needs. It serves as a structural component of macromolecules and acts as a cofactor for several essential cuproenzymes. Cellular respiration, free radical scavenging, iron metabolism, collagen synthesis, and numerous other vital functions in animal metabolism and health rely on copper (Linder, 2020). Deficiency in copper adversely affects performance metrics and immune system functionality (da Cruz Ferreira Júnior et al., 2022; Deo et al., 2023; Groff-Urayama et al., 2023).

Supplementing animal diets with copper beyond the basic nutritional requirements is a common practice to prevent deficiency symptoms and enhance growth across various species (Creech et al., 2004; Forouzandeh et al., 2022; Olukosi et al., 2018). Copper sulfate (CuSO4) is the most widely used copper supplement in animal nutrition due to its cost-efficiency and broad availability (Forouzandeh et al., 2022; Pang and Applegate, 2007; Park and Kim, 2016). Alternative inorganic sources, such as copper oxides and carbonates, are also utilized. However, these sources often suffer from lower bioavailability (da Cruz Ferreira Júnior et al., 2022; Mion, et al., 2023). As copper supplementation levels rise, the efficiency of its absorption generally diminishes, resulting in increased excretion of unabsorbed copper in feces, which could contribute to environmental contamination of soil and water resources, potentially harming biodiversity and ecosystems (da Cruz Ferreira Júnior et al., 2022; Ma et al., 2020; Vieira et al., 2020). In contrast, the utilization of organic copper sources, such as copper amino acid chelate and copper proteinate, might alleviate these issues. Organic minerals can be included at lower dietary concentrations compared to inorganic sources while achieving comparable or superior growth-promoting and health-maintaining effects, reducing mineral excretion (Ma et al., 2020; Świątkiewicz et al., 2014; Vieira et al., 2020).

Regarding the comparative effectiveness of organic and inorganic copper sources, it is generally posited that organic copper may exhibit higher bioavailability (Kong et al., 2022; Mion et al., 2023; Pino and Heinrichs, 2016). However, other studies have reported similar or even contrasting solubility levels between these sources. For instance, Clarkson et al. (Clarkson et al., 2021) observed comparable solubility percentages for sulfate and proteinate salt in deionized water and rumen fluid. Deters et al. (2021) found 100% solubility for CuSO4 in deionized water and hydrochloric acid (at pH 5.2 and pH 2.1, respectively), while bis-glycinate bound Cu exhibited solubility percentages of 68.9% and 80.6% in the same solutions. Moreover, based on regression analysis of liver or plasma Cu concentrations against total Cu intake by steers, copper sulfate demonstrated 100% relative bioavailability compared to 82% and 95% for bis-glycinate copper (Deters et al., 2021).

Therefore, this study aims to employ in vitro digestion models to evaluate the bioaccessibility of copper sulfate, copper amino acid chelate, and copper proteinate. Bioaccessibility, a key parameter in in vitro digestion research, refers to the proportion of a bioavailable substance released from the food or feed matrix into the gastrointestinal tract, making it accessible for intestinal absorption (Ayyash et al., 2021; Cao et al., 2021; Moreda-Piñeiro et al., 2012; Hur et al., 2011; Ting et al., 2015). Investigating bioaccessibility provides essential insights into the chemical transformations that different copper salts undergo within the digestive tract, thereby laying the groundwork for understanding their subsequent absorption.

MATERIALS AND METHODS

Materials

Copper amino acid chelate (Availa Cu, Cu-AA) was procured from Zinpro Corp., Shanghai, China, while copper proteinate (Bioplex Copper, Cu-P) was sourced from Alltech, Beijing, China. Copper (II) sulfate-5-hydrate (99%–102%), α-amylase (from Hog pancreas, powder, −50 U/mg), pepsin (from porcine gastric mucosa, p7000, ≥250 units/mg solid), and pancreatin (from porcine, p1750, meeting 4 × USP specifications) were acquired from Sigma-Aldrich. Hydrogen peroxide (30%) and nitric acid (guaranteed-reagent grade) were supplied by Aladdin. Dialysis bags with molecular weight cut-off values ranging from 0.1 to 1000 kDa were obtained from Spectrum Laboratories, Inc. (California). Fresh porcine bile was harvested from gall bladders and stored at −20°C until use. Other chemical reagents, meeting or surpassing analytical-reagent grade, were sourced from Sinopharm Chemical Reagent Co., Ltd. Ultra-pure water was obtained from a Milli-Q system (Thermo Scientific NANOpure).

Sample Preparation

Simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared at a concentration of 1.25× using the method outlined by Minekus et al. (Minekus et al., 2014). SSF comprised 15.1 mM KCl, 3.7 mM KH2PO4, 13.6 mM NaHCO3, 0.15 mM MgCl2(H2O)6, and 0.06 mM (NH4)2CO3. SGF consisted of 6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 47.2 mM NaCl, 0.1 mM MgCl2(H2O)6, and 0.5 mM (NH4)2CO3. SIF comprised 6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl, and 0.33 mM MgCl2(H2O)6. Adjustments to pH were made separately, using 1 M NaOH or 6 M HCl solution to achieve pH 7 for SSF, pH 3 for SGF, and pH 7 for SIF. The electrolyte stock solution was supplemented to achieve a 1× concentration before digestion. Purified diet was purchased from Trophic Animal Feed High-tech Co., Ltd, China, the composition was shown in Table 1.Table 1 Ingredient composition of purified diet.

Table 1Ingredient	g/kg diet	
Cornstarch	397.486	
Casein (>85% protein)	200.000	
Dextrinized cornstarch (90–94% tetrasaccharides)	132.000	
Sucrose	100.000	
Soybean oil (no additives)	70.000	
Fiber	50.000	
Mineral mix without Cua	35.000	
Vitamin mixa	10.000	
L-Cystine	3.000	
Choline bitartrate (41.1% choline)	2.500	
Tert-butylhydroquinone TBHQ	0.014	
a Copper was not added to the feed; whereas other vitamins and minerals were included in following the AIN-93 purified diets for laboratory rodents.

In vitro Digestion Procedure

The in vitro digestion models were established according to Minekus et al. (2014) with slight modifications (Figure 1). Salivary amylase was present at a concentration of 75 U/mL in SSF, while pepsin (3.2 g/L in SGF) and pancreatin (3.2 g/L) concentrations were determined according to Liu et al. (2015). Bile salt concentration, set at 10 mmol/L in SIF, was quantified using commercially available assay kits from the Jiancheng Institute, Nanjing, China.Figure 1 Flowchart of the in vitro digestion method. Three different forms of copper salts (Cu-sulfate, Cu-amino acid chelate and Cu-proteinate) are added individually to various diets (water, purified diet, corn starch and whey protein). The concentration of copper in the diets is 30 mg/kg. The concentration of salivary amylase is 75 U/mL in SSF, pepsin is present at a concentration of 3.2 g/L in SGF, while pancreatin and bile salt are both present at concentrations of 3.2 g/L and 10 mmol/L, respectively, in SIF. SSF, simulated salivary fluid; SGF, simulated gastric fluid; SIF, simulated intestinal fluid.

Figure 1

The in vitro digestion was conducted using a pH-stat (Titrando 902 and 800 Dosino, Metrohm, Herisau, Switzerland), with temperature control and mechanical mixing provided by a constant temperature water bath apparatus (HH601, Guohua Electronic Appliance Co., Ltd., Changzhou, China) and a magnetic stirrer (IKA, Staufen, Germany), respectively.

Certain copper salts (copper sulfate, Cu-AA, and Cu-P) were separately added to water, purified diet, whey protein, and corn starch to achieve a copper concentration of 30 mg/kg. Subsequently, 18 mL of SSF was added to the sample mixture and maintained at 37°C for 10 min. This was followed by the addition of CaCl2 and salivary amylase dissolved in 2 mL SSF, resulting in concentrations of 0.75 mM for calcium and 75 U/mL for salivary amylase. After 2 min of mixing, prewarmed SGF (40 mL, 37°C), premixed with an electrolyte stock solution, CaCl2, and pepsin, was added. The pH was adjusted to 3.0 using HCl, and the mixture was subjected to a 2-h digestion period. Subsequently, prewarmed SIF (80 mL, 37°C), premixed with an electrolyte stock solution, CaCl2, pancreatin, and bile, was added. The pH was then adjusted to 7.0 using NaOH solution (Figure 1).

For the control group, different copper salts were added separately to 80 mL of 0.1 M NaNO3, and the solution was carefully adjusted to pH 7.0. Temperature control (37°C) and mechanical mixing were maintained using the constant temperature water bath apparatus and magnetic stirrer, respectively.

At the onset of simulated intestinal digestion, a dialysis bag (10 kDa) containing simulated digestion fluid—comprising a mixture of SSF, SGF, and SIF—was submerged in digestive juices. Following 120 min of intestinal digestion, the bag was extracted. The extraneous digesta was utilized for determining the total copper concentration, while the solution within the bag was used for dialyzed copper determination. Total copper determination involved microwave-assisted acid digestion (Anton Paar Multiwave 3000), with nitric acid (4 mL) and hydrogen peroxide (2 mL) added to the digesta (2.5 mL). The solution from the dialysis bag was then diluted tenfold with ultra-pure water. For the control group, a dialysis bag (10 kDa) filled with 0.1 M sodium nitrate underwent dialysis in a copper solution for 120 min.

Copper concentration was assessed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Agilent Technologies, Santa Clara, CA, USA). Quality control samples were analyzed after every 5 samples. Calibrations were based on 2.0 mol/L nitric acid. The bioaccessibility, expressed as a percentage, was calculated using Equation 1.(1) Bioaccessibility(%)=Dialysedcoppper(10kDa,μg/l)Totalcopper(μg/L)×100%

Furthermore, in investigating the molecular size distribution of various copper salts, the digestion procedure was reiterated under consistent conditions. Distinct dialysis bags featuring varying pore sizes (0.1, 1, 100, and 1,000 kDa) were individually introduced into the solution. The overall copper content and the dialysed copper were quantified following established protocols. The addition of copper salts was carefully controlled to maintain accurate total copper concentrations. Any experimental data exhibiting a deviation in total copper concentration exceeding 15% were excluded. Control groups underwent analysis utilizing membranes of identical molecular weight cutoffs. The distribution, articulated as a percentage, was computed using Equation 2.(2) (M-N)Dialysisratio(%)=[Dialysedcoppper(N)−Dialysedcoppper(M)](μg/L)Totalcopper(μg/L)×100%

(M) and (N) represent the different sizes of the dialysis membranes.

Statistical Analysis

Each experiment was repeated three times independently, and the data are presented as mean ± standard deviation (SD). The study included three experimental groups: Group A (copper sulfate), Group B (copper amino acid chelate), and Group C (copper proteinate), each supplemented at a concentration of 30 mg Cu/kg diet. IBM SPSS Statistics version 22.0 (IBM Corp, Armonk, NY) was used for one-way analysis of variance (ANOVA). Post hoc comparisons were performed using the Least significant difference (LSD) test, with P < 0.05 indicating significant differences.

RESULTS AND DISCUSSION

In vitro digestion, employing enzymes like α-amylase, pepsin, pancreatin, and bile salts to mimic salivary and gastrointestinal digestion, aid in predicting in vivo outcomes by assessing nutrient metabolism in the digestive tract (Cao et al., 2021; Lima et al., 2014; de Oliveira Gonçalves et al., 2020). This study assesses the in vitro bioaccessibility of different copper salts across sodium nitrate solution (without digestive enzymes), water, purified diet, whey protein, and corn starch. Despite variations in digestion processes among animal species, a commonality lies in the acidic stomach, neutral intestines, and requisite digestive fluid levels. Additionally, mineral absorption predominantly occurs in the small intestine, underscoring the importance of tracking mineral transformations from the acidic stomach to the neutral gut for comprehending their bioavailability (Silva et al., 2020). Therefore, employing a continuous in vitro digestion model is pivotal for investigating mineral bioaccessibility in organisms.

During the study, it became apparent that the morphological alterations of different copper salts in the digestive tract are influenced by various factors. In addition to transformations experienced by copper salts due to variations in digestive pH and enzyme activity, the digestion process itself alters the composition, quantity, and spatial arrangement of substances within the digesta. These collective factors synergistically influence the chemical form of copper within the digestive tract, thereby impacting its bioaccessibility.

After a 2-h dialysis in water, it is evident that the bioaccessibility of copper amino acid chelate is markedly higher (P < 0.05) compared to copper sulfate and copper proteinate (Figure 2A) in nitric acid solution. Copper sulfate, despite its high-water solubility, exhibits low concentrations of dissolved copper ions (<0.1 mg/L at pH 7.0) due to its relatively small solubility constant (2.2 × 10−20 at 25 ℃) (Speight, 2005), leading to reduced bioaccessibility (Figure 2A, 34% ± 1%). In the absence of complexing ligands, a large amount of copper precipitates as copper hydroxide in neutral environments, thereby decreasing bioaccessibility. This effect is illustrated in Figure 3A, where the proportion of copper exceeding 1,000 kDa is as high as 55% ± 5%. Copper proteinate exhibits relatively low bioaccessibility (36% ± 4%) under same condition (Figure 2A). From Figure 3A, it evident that a significant portion of copper proteinate is distributed above 1,000 kDa. This distribution suggests that the increased molecular weight of copper complex ligands (peptides or proteins) or polymerization phenomena among these ligands at neutral pH hindering the passage of molecules exceeding 10 kDa. On the other hand, copper amino acid chelate form complexes with amino acids characterized by their small molecular weights, facilitating the passage of a 10 kDa dialysis membrane in nitric acid solution, resulting in substantially higher bioaccessibility compared to copper sulfate and copper proteinate (Figure 2A). It can be observed from Figure 3A that 55% ± 5% of amino acid copper chelate is distributed between 0.1 and 1 kDa.Figure 2 In vitro bioaccessibility of CuSO4, CuAA, and CuP in control group (A), water with digestive enzyme (B), purified diet (C), corn starch (D), and whey protein (E). Values are shown as the mean ± standard deviation (SDs), n = 3, **P < 0.05. CuAA, Cu-amino acid chelate; CuP, Cu-proteinate.

Figure 2

Figure 3 Molecular size distribution of Cu-sulfate (CuSO4), Cu-amino acid chelate (CuAA) and Cu-proteinate (CuP) in control group (A), water with digestive enzyme (B), purified diet (C), corn starch (D), and whey protein (E).

Figure 3

No significant difference (P > 0.05) in the bioaccessibility among the three types of copper was noted after the addition of digestive enzymes (Figure 2B). For copper sulfate, the majority of copper is distributed between 0.1 and 1 kDa (60% ± 4%, Figure 3B). The addition of digestive enzymes and the subsequent degradation of proteins (enzymes) during the in vitro digestion process may introduce organic ligands, which can bind to copper, thereby reducing the formation of copper hydroxide. When copper binds to these small organic ligands, the resulting complexes, with lower molecular weights, facilitates their passage through a 10 kDa dialysis membrane, thereby increasing the bioaccessibility of copper sulfate (Figure 2B, 81% ± 2%). The distribution of copper proteinate is relatively high in the range of 0.1 to 1 kDa and 1 to 10 kDa, respectively (Figure 3B). Copper proteinate has already formed a complex structure with proteins and peptides, the increase in its bioaccessibility can be attributed to the presence of digestive enzymes, which lead to the degradation of protein ligands, reducing the volume of the complex and thereby enhancing bioaccessibility (Figure 2B, 81% ± 4%).

After the addition of purified diet or whey protein, the bioaccessibility of copper amino acid chelate and copper proteinate was significantly higher than that of copper sulfate (Figure 2C and E, P < 0.05). In the purified diet system, the distribution of copper between 10 and 100 kDa increased across all copper salts. Copper sulfate exhibited the most pronounced increase, from 12% ± 11% to 48 % ± 7%, while copper amino acid chelate and copper proteinate increased from 9 % ± 2% to 27 % ± 1 % and 12% ± 6 % to 24 % ± 8 %, respectively (Figure 3C). Similarly, in the whey protein system, copper distribution between 10 and 100 kDa is raised to 30 % ± 12% for copper sulfate, 17% ± 9% for copper amino acid chelate, and 13 % ± 4% for copper proteinate (Figure 3E). The molecular size distribution suggests the presence of strong copper complexes in both the purified diet and whey protein, likely derived from casein and whey proteins. These proteins, along with their inherent functional groups, competitively chelate with copper, thereby increasing the volume of the copper complex and reducing copper bioaccessibility. Conversely, copper amino acid chelate and copper proteinate, being inherently bound with organic compounds, exhibit competitive advantages, resulting in higher bioaccessibility compared to copper sulfate (P < 0.05).

Investigating the starch system revealed no significant difference in bioaccessibility among the three copper sources (Figure 2D, P > 0.05). Analysis of copper distribution revealed a decrease in the proportion distributed between 1 and 10 kDa, accompanied by an increase between 10 and 100 kDa. However, copper salts with a molecular volume of 0.1 to 1 kDa experience a relatively smaller decline (Figure 3D). Starch hydrolysis products, such as sucrose and glucose, demonstrate lower chelating capacity for copper compared to amino acids and proteins (Wu et al., 2021). The extended chain structure of starch likely creates molecular steric hindrance, impeding the passage of copper salts through the dialysis membrane and consequently reducing their bioaccessibility. Conversely, copper salts with a molecular volume of 0.1 to 1 kDa encounter comparatively less hindrance due to their smaller molecular size.

Experimental findings highlight a notable superiority in the bioaccessibility of organic copper over copper sulfate, particularly evident in both the feed model and whey protein model. The high content of copper sulfate tends to precipitate as copper hydroxide under neutral and alkaline conditions, hindering copper absorption. However, the presence of feed constituents mitigates the formation of copper hydroxide in the digestive tract. Organic copper demonstrates the capability to mitigate copper's complexation with other dietary constituents in the digestive tract, thereby enhancing its bioaccessibility. Proteases secreted during digestion can hydrolyze the ligands of copper proteinate, thereby reducing its molecular weight. In certain scenarios, the bioaccessibility of copper sulfate is significantly inferior to that of organic copper.

Analyzing identical copper salts across varied diets reveals significant variations in their bioaccessibility (Figure 4). For copper sulfate (30 mg/kg, Figure 4A), copper tend to precipitate as copper hydroxide in sodium nitrate solution due to the rising concentration of hydroxide ions. However, concurrent ingestion of enzymes or feed introduces organic ligands, thereby enhancing copper solubility. The bioaccessibility of copper correlates with the molecular weight of these ligands. When only digestive enzymes are present, digestion is thorough during the in vitro digestion system, resulting in the breakdown of organic matter into smaller molecules. Conversely, the introduction of feed leads to more larger molecules, prompting copper to form complexes with various organic ligands dictated by their complexation constants. Consequently, some copper forms complexes with larger molecules, thereby diminishing the bioaccessibility of copper sulfate.Figure 4 In vitro bioaccessibility of CuSO4 (A), CuAA (B) and CuP (C) in water (control group), water with digestive enzyme, purified diet, corn starch, and whey protein. Values are shown as the mean ± standard deviation (SDs), n = 3, ** P < 0.05. CuAA, Cu-amino acid chelate; CuP, Cu-proteinate.

Figure 4

The presence of digestive enzymes also enhances bioaccessibility for copper amino acid chelate (Figure 4B). Perhaps a small number of macromolecular proteins present in copper amino acid chelated are effectively reduced in molecular weight by enzyme digestion, thereby augmenting bioaccessibility. Notably, compared to digestive enzymes and whey protein systems, the bioaccessibility of copper amino acid chelate is lower in purified diet and corn starch systems (P < 0.05). In the purified diet system, this discrepancy is primarily attributed to the molecular weight of casein, which presents greater resistance to digestion compared to whey protein. During digestion, some copper dissociates from amino acids and forms complexes with various ligands coexisting in the digestive tract, resulting in complexes of different molecular sizes. In corn starch systems, the steric hindrance of starch prevents copper from passing through the simulated intestinal tract (semi permeable membrane), thereby resulting in reduced bioaccessibility.

The bioaccessibility of copper proteinate shows consistent fluctuations across various dietary contexts. The presence of digestive enzymes facilitates the hydrolysis of protein ligands within copper proteinate, thereby decreasing its molecular weight and consequently enhancing its bioaccessibility. The presence of large molecules and indigestible substances within the diet contributes to a decrease in the bioaccessibility of copper proteinate, resembling the patterns observed in copper amino acid chelate.

The simultaneous consumption of feed ingredients significantly influences the bioaccessibility of copper salts. Digestion at lower protein concentrations results in faster degradation rates, yielding smaller organic ligands (Zhang and Vardhanabhuti, 2014). Consequently, in the presence of only digestive enzymes, all three copper salts exhibit higher bioaccessibility. However, with the introduction of whey protein and purified diet, incomplete protein digestion leads to the formation of macromolecular proteins complex with copper, thereby reducing bioaccessibility. The presence of long-chain macromolecules in the diet substantially diminishes the bioaccessibility of all three types of copper, potentially attenuating the advantages of organic copper. Thus, organic copper may not consistently exhibit higher bioaccessibility than inorganic copper in all systems.

Our research indicates that the digestion process significantly influences the bioaccessibility of copper salts, leading to potential variations in bioavailability across different animal species. Variations in digestive characteristics, such as rumen environment, digestive duration, and composition of digestive juice, can induce distinct chemical transformations in copper. Previous studies investigating the solubility of different copper sources have reported consistent percentages of soluble copper for sulfate and proteinate salts in both deionized water and rumen fluid (pH 6.2), which contrasts with our findings (Clarkson et al., 2021). Deters et al. observed 100% solubility for CuSO4, compared to 68.9% and 80.6% for bis-glycinate bound Cu in deionized water and hydrochloric acid (pH 5.2 and pH 2.1, respectively) (Deters et al., 2021). The substantial disparities in these findings from ours primarily stem from variations in the concentration of the chosen copper salt and pH levels. Hence, when assessing the bioaccessibility of copper salts across diverse animal species, factors such as the intestinal pH, digestive duration, the concentration of copper in the diet, and chemical composition must be considered.

In addition to considering the bioaccessibility of different copper salts, various transport pathways may exist for distinct forms of copper within intestinal epithelial cells, including high-affinity Cu(I) transport mediated by Ctr1 and Ctr1-independent Cu (II) transport (Lee et al., 2002; Tsang et al., 2021). Our previous study have identified divergent absorption pathways for complex copper and ionic copper in Caco-2 cells, resulting in differences in absorption efficiency (Wu et al., 2023). Gao et al. (Gao et al., 2014)reported significantly higher apparent permeabilities of copper in amino acid complex forms compared to CuSO4 in Caco-2 cells. Further research is needed to elucidate potential disparities in transport pathways for organic and inorganic copper in the intestine, which may contribute to differential physiological effects. Moreover, the interplay among rumen microorganisms (Faulkner et al., 2017; Kong et al., 2022; Mion et al., 2023), minerals (Lee et al., 2021), copper potential toxic effects on rumen microbes and fermentation processes (Lei et al., 2023; Pino and Heinrichs, 2016), and other substances (Broom et al., 2021; Vigh et al., 2023) can also influence mineral absorption outcomes.

In conclusion, the study utilized an in vitro digestion model to assess the bioaccessibility of three distinct copper types. Results indicates that elevated concentrations of copper sulfate could lead to the precipitations of copper hydroxide in the intestines under neutral conditions when no organic ligands were present. Conversely, organic copper demonstrated superior bioaccessibility under similar circumstances. Feed serves a dual purpose in copper metabolism: providing organic ligands to prevent copper hydroxide formation while competitively chelating with copper ions. Hence, when selecting copper salts, it is crucial to consider both the dosage of copper salts and the composition of the diet comprehensively.

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS

This research was supported by the the Foundation of Zhejiang Provincial Key Laboratory of Food Safety (1110KZN0416098G ).
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