Roles of zinc in the gut-liver axis

Abstract

The gut and the liver are the main organs in the regulation and distribution of zinc. Therefore, gut and liver disease impact zinc functions in other organs. Many of the phenomenological observations made in the past century concerning the role of zinc in growth and development and the role of zinc deficiency in many diseases are now better understood on the basis of zinc’s remarkable catalytic, structural, and regulatory functions in over 3200 human proteins and its functions as an ionic messenger similar to calcium in intra- and extracellular communication, regulation of metabolism, and gene expression. Zinc has key roles in carbohydrate and lipid metabolism, nitrogen balance, pH control, and the synthesis and degradation of proteins. Its classification as a trace element distracts from its global significance in the proliferation and differentiation of all cells. Zinc is at least as important as iron, if not even more so. Its intricate cellular regulation by 24 membrane zinc transporters, a dozen metallothioneins and other zinc homeostatic proteins supports this tenet. This review will summarize the role of zinc in the integrity of the intestinal barrier, in maintaining a healthy gut, and, through the gut-liver axis, a healthy liver. Zinc is critical for a proper immune response to support and control inflammation, in fighting off insults and repairing tissues, but also in avoiding chronic inflammation. About 75% of patients with decompensated liver cirrhosis are zinc deficient. Zinc deficiency, a prooxidant and proinflammatory condition, needs clinical attention in liver disease, should include attention to gut health, and involve pharmacological treatment with supplemental zinc. Monotherapy with zinc alone, however, is not the answer. Along with zinc, additional therapeutics are required to restore intestinal and hepatic functions.

1

Introduction

The liver and the gut have central roles in controlling systemic zinc homeostasis and thus functions of zinc. Accordingly, there is a strong relationship between the functions of zinc and the functions of the liver and the gut. Zinc deficiency compromises hepatic functions and is often observed in liver disease [1,2]. The gut supplies the liver with nutrients including zinc through the portal vein, and therefore gut health is important not only for the proper nutrient supply to the liver but also for avoiding leakage of potentially liver-damaging substances when gut integrity is compromised. Importantly, zinc also has a critical role in gut health and barrier functions. This relationship between the gut and the liver is referred to as the gut-liver axis. Furthermore, the liver is the central organ in other gut-organ axes such as the gut-brain or the gut-skin axis, because these axes involve passage of compounds through the liver or metabolism in the liver. In fact, these gut-liver-organ axes are not only tripartite but tetrapartite as they include the microbiome, i.e., they are microbiome-gut-liver-organ axes (Fig. 1). In this article, we will focus on the role of zinc in the central segment, the gut-liver axis, and the crosstalk between these organs in the regulation of the metabolic and inflammatory processes through host-microbiome interactions [3].

Fig. 1.

The gut-liver axis (red) is the central segment in any other gut-organ axis and in the control and distribution of zinc. Assessment of zinc status and determining the factors that determine zinc uptake are critical for health and need to be monitored in liver and gut disease.

A healthy gut microbiome includes interactions of a variety of microorganisms (bacteria, fungi (yeast), and viruses). An impressive demonstration of the positive effect of the communication of the microbiome with the liver via the gut is the observation that a fungal secondary metabolite affects gut ceramide metabolism and thus ceramide signaling to the liver with the consequence of improving metabolic dysfunction-associated steatohepatitis (MASH) [4]. In a healthy human, commensal bacteria participate in the defense against pathogenic bacteria and their potentially toxic products. The host is also defending itself against pathogenic bacteria by secreting antimicrobial metabolites and peptides [5]. In addition, the gut microbiome influences different metabolic processes of the host, affects the integrity of the mucosal and epithelial cell barriers of the gut, and the development, homeostasis and functions of the innate and adaptive immunity [5]. The gut barrier is a highly evolved structure of a complex network of structural, biochemical, and immunological components to protect the host from pathogenic bacteria and toxic products that would otherwise enter the systemic circulation [6]. The gut and the liver communicate via the portal vein, the hepatobiliary system, and indirectly via mediators in the systemic circulation through the hepatic artery and vein, together referred to as the gut-liver axis. With the unique anatomy and the vascular system of the gut-liver axis, the liver receives approximately 70 % of its blood supply from the intestine via the portal vein [7]. Through their metabolic products and transformations of substrates transported in the portal vein, microbes in the intestine are involved in the maintenance of liver homeostasis. However, alterations in the normal composition and diversity of the gut microbiome – so-called dysbiosis - can be a source of pathogens and molecules that contribute to the onset of liver disease or progression of chronic liver disease. The portal vein feeding into the liver contains microbes and their products such as endotoxins, and bacterial DNA [8], which need to be controlled. One additional control takes place at the liver sinusoids through the liver sinusoidal endothelial cell (LSEC) barrier, which undergoes phenotypic changes in liver disease [9,10]. Portal blood also contains bacterially produced metabolites such as ethanol, trimethylamine, BA, and short chain fatty acids, some of which have beneficial effects. Thus, an increased intestinal permeability and dysbiosis impact metabolic pathways and inflammatory processes in the liver [8]. With its various capacities of detoxification, biotransformation, secretion, and its immunological competence, the healthy liver has special importance in fighting harmful substances to which our body is exposed. The concept of a “leaky gut” means that the gut barrier is compromised, and the gut becomes permeable to bacteria, fungi, and viruses, their products, and other compounds in our diet, with serious consequences for the liver. A leaky gut is an important pathogenic factor for developing liver disease and life-threatening complications in liver cirrhosis, such as hepatic encephalopathy, ascites, and infections that can lead to septicemia [8,11]. An increased gut permeability also contributes to the pathogenesis of earlier stages of inflammatory liver disease such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) [12,13] and possibly fibrosis of chronic hepatitis C [14,15]. Thus, microbial dysbiosis together with increased gut inflammation and a weakened immunological response of the liver have a detrimental role in the development of NASH, ASH, and subsequent compensated and decompensated cirrhosis that can progress to liver cancer (hepatocellular carcinoma, HCC) [16–19] (Fig. 2). Systemic inflammation plays a crucial role in disease progression of cirrhosis and is a key driver for acute decompensation and thus the progression from compensated to decompensated cirrhosis [20].

Fig. 2.

The different stages of liver disease and the gut-liver axis, including the microbiome. The extent of zinc deficiency increases with progression of liver disease. The gut is the main organ for zinc absorption and secretion. From the gut zinc is transferred to the liver via the portal vein, distributed to other organs, but also a surplus returned to the gut via the bile and the mesenteric artery in the enterohepatic circulation. The microbiome has a major role in determining the integrity of the gut barrier and the uptake of zinc.

In 2023, a new nomenclature for steatotic liver diseases (SLD) was introduced in a consensus-based process. MASLD and MASH (metabolic dysfunction-associated steatotic liver disease or steatohepatitis) were grouped under the term SLD in addition to alcohol-associated liver diseases, other causes of fatty liver and cryptogenic liver diseases [21,22]. MASLD and MASH replace the terms NAFLD and NASH. In contrast to the old definition of NAFLD, MASLD is defined by the detection of fatty liver disease and at least one of the cardiometabolic factors (insulin resistance, abdominal obesity, arterial hypertension, dyslipidemia, atherosclerosis).

This review provides an overview of our present knowledge of the role of zinc in human gut and liver health, specifically in the gut-liver axis, with an emphasis on the importance of maintaining a healthy zinc status.

2

Zinc: physiological and biochemical functions and significance for health

Zinc is an essential micronutrient for all living organisms and controls proliferation, differentiation, and apoptosis of cells through its fundamental roles in multiple metabolic processes, signal transduction, and the control of gene expression [23,24]. The plethora of its functions are based on intra- and extracellular actions and primarily interactions of Zn(II) ions with proteins [25,26]. Functions of zinc in proteins include catalysis in hundreds of enzymes, maintenance of protein structure, including the organization of protein domains for interactions with other proteins or other biomolecules, bridging proteins in protein-protein interactions, and regulatory functions in signal transduction and the control of gene expression. Zinc has much wider roles than the other trace metals iron, copper, and manganese together, a property that makes it a Type 2 nutrient with general functions, while iron, copper, and manganese are Type 1 nutrients with specific functions [27]. The number of functions of zinc are several orders of magnitude higher than those of some vitamins such as B12, B6, or K [28,29]. Although overall counts that over 3200 human proteins require zinc as a cofactor have been given repeatedly [30], the impact of either zinc deficiency or a perturbation of the cellular homeostasis of zinc on liver function and disease is rarely acknowledged in major review articles. There are multiple reasons for this omission. One reason is the absence of appropriate biomarkers for tissue zinc status and a lack of knowledge about which molecular processes are affected when zinc is deficient. In clinical chemistry, zinc is determined in the blood as a trace of a trace element as only 0.1 % of the total body zinc is in blood. In cells, however, the concentrations of zinc are as high as those of major metabolites. We do not know to which extent zinc deficiency in blood reflects a zinc deficiency in the liver or any other organ. For example, in the acute phase response in inflammation, an apparent zinc deficiency in blood is associated with an increase of zinc in the liver [31–33]. Increased cellular zinc leads to gene expression of a significant number of proteins and affects the function of proteins that otherwise are not targeted by zinc. A second reason is that we do not know whether all zinc-dependent processes are affected to the same extent when a zinc deficiency develops or zinc distribution is a hierarchical process in which some zinc-dependent functions are sacrificed if the deficiency is mild while others are affected only if the zinc deficiency becomes more severe. The recent discovery of a genuine zinc chaperone protein indicates a hierarchy, namely that preference is given to the allocation of zinc for maintaining some processes [34]. We do know, however, that zinc is essential for growth and the cell cycle and that there is cell cycle arrest if not enough zinc is available. Clearly, this process is important for tissue regeneration, because hepatic or intestinal cells cannot renew under severe zinc deficiency. The impact of zinc on molecular and cellular processes is enormous and includes functions in over 1200 enzymes of all seven classes, in about 1000 transcription factors, and another 1000 proteins with yet unknown functions [30]. To demonstrate the extent to which zinc is involved in cellular functions we have listed some key zinc enzymes/proteins involved in processes that are important in general and for gut or liver health in particular (Table 1).

Table 1.

Examples of human zinc enzymes/proteins in processes that are important for the gut and liver.

Enzyme/Protein (number of genes)	Type of Sitec: catalytics: structuralr: regulatory	Function/Process
Alcohol dehydrogenases [7]	c, s	involved in alcohol (ethanol)/aldehyde metabolism but also other substrates such as retinol, fatty acid alcohols, steroids, lipid peroxides, drug transformations; class III alcohol dehydrogenase is a reductase, i.e., an S-nitrosoglutathione reductase and a formaldehyde reductase through adduct formation with glutathione (GSH), S-nitrosothiol homeostasis; effect on NAD^+/NADH ratio
Sorbitol dehydrogenase	c	fructose metabolism; effect on NAD^+/NADH ratio, osmotic pressure
Carbonic anhydrases [14]	c	acid/base balance: CO2/HCO3^-, carbonic anhydrase Va is exclusively expressed in the liver; control of substrate utilization in carboxylations
Dihydroorotase	c	pyrimidine synthesis
δ-Aminolaevulinate dehydratase	c	porphyrin synthesis, heme enzymes, cytochromes, detoxification (P450)
Prot(ein)ases and peptidases	c, (s)	proteostasis, hormone processing, collagenases, progelatinases and matrix metalloproteinases/ADAMs - at least 23 expressed in humans (involved in fibrosis), some have also structural zinc sites, neprilysin (endopeptidase), carboxy- and aminopeptidases (pancreas), e.g., methionine aminopeptidase, sonic hedgehog, TACE (TNF-α-converting enzyme), ACE (angiotensin converting enzyme), glyoxalase
Adenosine deaminase [2]	c	purine metabolism, important for immune system
Adenosine deaminase acting on RNA (ADAR) [3]	c	RNA editing (role in the editome), adenosine to inosine conversion
APOBEC (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide), family of cytidine deaminases (several)	c	cytosine to uracil conversion in both DNA and RNA
Poly (ADP ribose) polymerase (several)	c, s	involved in DNA repair, programmed cell death, includes tankyrase, a poly (ADP-ribosyl) transferase, involved in the Wnt signaling pathway, control of telomere length and vesicle trafficking, the structural zinc sites are in zinc fingers
Cytosine methyl transferase [4]	c	DNA methyl transferase. Note: Zinc is required for the activity of various epigenetic enzymes, such as DNA methyltransferases (DNMTs),histone acetyltransferases (HATs), histone deacetylases (HDACs – see below), and histone demethylases, which possess several zinc binding sites.
Glutaminyl peptide cyclotransferase	c	a transferase that forms 5-oxoproline (pyroglutamic acid)
Methionine synthase	c	One-carbon metabolism
6-Pyruvoyl-tetrahydropterin synthase	c	tetrahydrobiopterin synthesis
Leukotriene A4 hydrolase	c	leukotriene B4 synthesis
GTP cyclohydrolase 1	c	GTP synthesis
Lys-specific demethylases (KDMs)	s	modifying histones and influencing gene expression, KDM2A and B have zinc finger domains that participate in DNA binding, JHDM (JmjC domain containing histone demethylases)
Farnesyl- [1] and geranylgeranyltransferases [1]	c	posttranslational modifications that are important for protein localization
Palmitoyl acyltransferase (PATs) [23]	s	zinc in the Cys-rich domain, also serving as a membrane-anchor
Ubiquitin E3 ligases (>600)	s	RING finger E3 ligases are the largest group, protein degradation (proteostasis)
SUMO transferases (E3 ligases) [4]	s	adding SUMO (small ubiquitin-like) modifier to proteins for stability, localization, and interaction with other proteins (sumoylation) ZNF451
Histone deacetylases	c	epigenetic modifications
Phosphatases, esterases	c	alkaline phosphatase, phospholipase C, phosphodiesterase
Aspartate transcarbamoylase	s	urea cycle, acid/base balance: NH3/NH4^+
Cytochrome c oxidase	s	mitochondrial respiratory chain
Superoxide dismutase [2]	s	binuclear zinc/copper site in cytosolic and extracellular enzyme
Nitric oxide synthase	s	binding between subunits in the dimer
Kinases	s	protein kinase C, Bruton’s tyrosine kinase, casein kinase2β, diacylglycerol (DAG) kinase, thymidine kinase, pyridoxine kinase, S6 kinase, raf-1, protein tyrosine kinase
Phosphatases	c	Protein phosphatase 1, pyrophosphatase
Transcription factors	s	protein-protein, protein-DNA interactions in controlling gene expression, and protein-RNA interactions, e.g., nuclear hormone receptor familyFXR (receptor for FGF19/21), PXR (receptor for pregnane), retinoid receptors, glucocorticoid and sex hormone receptors, peroxisome proliferator-activated receptors (PPARs), glucocorticoid,HNF4 in alcoholic liver diseaseBRCAs
Ribosome	s	various zinc finger motifs, RNA polymerase subunit RBP10
Tripartite-motif proteins (TRIM) [75]	s	RING finger domain, pathogen recognition and host defense
Ubiquitin hydrolases, JAMM family [7]	s	DUBs: deubiquinating enzymes
Protein interface sites	s	Glyoxalase I, NOS, psoriasin (S100A7), interferon dimers, prolactin receptor/growth hormone, Apo2IL/TRAIL

ADH, alcohol dehydrogenases; GSH, glutathione; NADH, reduced nicotinamide adenine dinucleotide; NAD+, nicotinamide adenine dinucleotide.

The proteins include zinc enzymes involved in pH and redox control, control of some metabolic pathways as committing enzymes, proteostasis, the synthesis of some cofactors and vitamins, detoxification, posttranslational and epigenetic events, repair processes, and yet others. For structural zinc sites, the role of zinc includes protein-protein and protein-DNA/RNA interactions for many purposes. Moreover, the significance of zinc for cellular function is evident from the fact that at least 24 transporters of the ZIP (SLC39A) and ZnT (SLC30A) families control its cellular and subcellular homeostasis and distribution in addition to about a dozen metallothioneins (MTs) that buffer the available zinc ions dynamically, zinc sensing transcription factors that respond to fluctuations of zinc ions and control gene expression, and chaperone proteins that allocate zinc ions to some proteins (Table 2, Fig. 3).

Table 2.

Cellular zinc homeostatic proteins.

Protein Family	Proteins	Role in Zinc Homeostasis	Function/Localization
ZIP transporters (SLC39A),14 members	ZIP1–14	Importers	Increase cytosolic zinc by transporting zinc into cells from the extracellular space or internal organelles (Zip7); ZIP14 mediates hepatic zinc uptake during inflammation; localized to the plasma membrane or intracellular organelles, variation of expression in different cells
ZnT transporters (SLC30A), 10 members	ZnT1–10	Exporters	Decrease cytosolic zinc by transporting zinc out of cells (ZnT1) or into intracellular compartments such as exocytotic vesicles (Zn2,3,8), Golgi apparatus, or secretory vesicles, ZnT9 is a mitochondrial zinc exporter, variation of expression in different cells
Metallothioneins	MT-1 (at least 8 human genes), MT-2, MT-3, MT-4	Buffers	Buffer free zinc ions dynamically; involved in redox-regulated zinc release and oxidative stress protection; variation of expression in different cells
MTF-1	Metal regulatoryTranscriptionFactor	Sensor	Senses elevated zinc concentrations, translocates to nucleus and activates expression of specific genes
ZNG1	Zn-regulated GTPase metalloprotein activator	Chaperone	Transfers zinc to methionine aminopeptidase 1, coupled to GTP hydrolysis

Fig. 3.

Simplified view of cellular zinc (Zn) distribution. The figure shows half a cell with a mitochondrion (top) and the subcellular distribution of intracellular zinc ([Zn²⁺]_i) to other organelles in the same cell (bottom). The expression levels of Zip and ZnT transporters and metallothioneins differ in different cells. Vesicular exocytosis of Zn is employed by some cells only. For human liver, RNA levels of Zip and ZnT transporters have been reported [35]: Zip9>Zip3,6,7,8,11,14>Zip2,10,13 and ZnT10>ZnT5,6,7,9>ZnT1,4. The expression levels of the different metallothioneins change during the development of liver disease [36]. MTF-1: metal regulatory transcription factor-1. The figure was generated with the use of Servier Medical Art.

The exquisite system of this cellular homeostatic control is evident when compared to iron, for example, where only one cellular importer (DMT1) and one exporter (ferroportin) exist in addition to transferrin receptor-mediated endocytosis. Any perturbation of the homeostatic control of zinc leading to either too low or too high cellular zinc concentrations has severe consequences. Environmental, nutritional and genetic factors can affect the functions of these zinc homeostatic proteins and perturb the system. Examples for genetic variations are mutations in the main zinc uptake transporter ZIP4 in the gut, which can lead to life-threatening zinc deficiency in acrodermatitis enteropathica if not treated with life-long zinc supplementation. Numerous other mutations in zinc transporters are associated with many diseases, including effects on the gut-liver axis [37]. For example, a rare loss-of-function mutation in ZIP5, which is located on the basolateral side of intestinal cells, results in increased zinc in the blood and improved liver functions in obese individuals, suggesting that ZIP5 is a therapeutic target in NAFLD [38]. Vice versa, a genetic condition that leads to high serum zinc (hyperzincaemia) and hypercalprotectinaemia presents with hepatosplenomegaly [39]. Zinc controls liver health through the regulation of the zinc-dependent gene expression of zinc transporters and MTs. Human liver samples from patients with MASH have less MT-1B protein than those from healthy subjects [40]. This finding regarding MT was also observed in mice fed a high-fat diet and in a hepatocyte cell line grown in a medium containing free fatty acids. Further exploration of this observation demonstrated that zinc-induced MT-1B has a role in attenuating hepatic inflammation and steatosis by inhibiting the PI3K/AKT signaling pathway [40]. If homeostatic control does not maintain the concentrations of zinc and other essential metal ions in a physiological range, a phenomenon referred to as dyshomeostasis, mismetalation of a metalloprotein with the wrong metal ion can occur. For example, at low zinc concentrations, other metal ions can bind at zinc sites, including toxic ones such as cadmium, and compromise the functions of the corresponding proteins. At high zinc concentrations, zinc can compete at metal sites of other metalloproteins. Furthermore, it is now well documented that zinc ions are intracellular and intercellular messengers [41]. The intercellular functions include autocrine, paracrine, and endocrine effects. While the number of targets of these signaling zinc ions is unknown, it is not insignificant. For example, in one signaling pathway alone, the TNF/NF-κB signaling pathway, there are at least 33 zinc-protein interactions [42]. Remarkably, zinc ion signaling adds many more proteins that bind zinc transiently to the already impressive count of over 3200 bona fide zinc metalloproteins. Finally, the control of zinc is linked to the control of other metal ions such as copper and iron, causing shifts in the usage of these metal ions with yet additional consequences for health if the control is compromised.

In addition to the crosstalk of zinc with other metal ions, an important interaction is with redox biology. Zinc influences redox metabolism although it is redox-inert itself [41]. Its functions are described as pro-antioxidant and anti-inflammatory. Zinc protects against oxidative distress, and this protection is lost in zinc deficiency, leading to lipid oxidation and DNA damage and eventually triggering cell death. Likewise, too high zinc concentrations can also have adverse effects on the redox balance. The relationship between zinc and redox metabolism is reciprocal. Conditions of oxidative distress release zinc ions from proteins and thus contribute to dyshomeostasis of zinc, including a loss of cellular zinc.

Regarding the gut and liver diseases discussed here, zinc is also essential for the control of inflammation, components of the innate and adaptive immunity, for haemostasis, and has a central role in all phases of wound healing [43,44]. Zinc deficiency results in a reduction of function and/or dysfunction of all immune cells [45]. Consequently, individuals with zinc deficiency have an increased risk for infections, unresolved inflammation, autoimmune diseases, and tumor development [45–47].

3

Zinc uptake in the gut: the gut-liver axis

The gut decides which nutrients are taken up and together with the kidney which substances are secreted. Gut health, and specifically the proper control of the gut’s barrier function and permeability, therefore, are critical for the health of other organs and the entire organism [48].

Nutrient uptake is not only a matter of the nature of the food itself but also the composition of the microbiome in the lumen of the gut. The microbiome includes bacteria, fungi (mycobiome), and viruses (microvirome) and is described as a supporting organ of the host. It is determined by environmental factors (diet, medication, lifestyle) and genetic and other factors of the host. Thus, the host controls the microbiome with biomolecules such as antimicrobial peptides (AMPs) made by specific intestinal epithelial and immune cells. The microbiome changes in the life course of the host.

Between the gut lumen with the microbiome and the gut surface is the mucus layer. To maximize nutrient uptake, the gut surface is folded with crypts and villi. The cell types in the gut are enterocytes, Goblet cells interspersed with enterocytes and producing mucus, Paneth cells that together with enterocytes fight infections and harmful bacteria with AMPs, enteroendocrine cells producing gastrointestinal hormones and communicating with the nervous and immune systems and the microbiome, and immune cells. Finally, there is innervation and vascularization of the gut, connection to capillaries of the portal vein and the lymphatic system, the lacteals, which are in the Peyer’s patches that include the gut-associated lymphoid tissues (GALT) and the mucosa-associated lymphoid tissue (MALT). The microfold cells (M cells) are responsible for mucosal immunity and are involved in the transport of microbes and substances across the epithelial cell layer to the lamina propria for interactions with immune cells. Zinc has general and specialized functions in all these cells and is thus an important factor for gut health [49,50].

Zinc is taken up from food at the brush border membrane of the enterocytes (intestinal epithelial cells) primarily in the small intestine [51]. The zinc transporter proteins involved in enterocytes are the main importer ZIP4, which is up- or downregulated depending on zinc status, ZnT5B, which is involved in bidirectional transport [52] on the apical (mucosal) side, the main exporter ZnT1 for secretion into the portal blood, and ZIP5 and 14 on the basolateral (serosal) side [53]. ZnT1 has a critical role in controlling systemic zinc homeostasis [54]. Several cation channel proteins of the TRP (transient receptor potential) family also have a role in zinc transport [55]. For example, the member TRPM7 has been identified as a gatekeeper of intestinal mineral absorption in mice, influencing the survival of offspring [56]. The G-protein coupled receptor GPR39, which is also called zinc receptor (ZnR) because it links fluctuations of extracellular zinc to calcium signaling in cells, regulates intestinal ion transport and barrier function in epithelial cells and has been implicated in modulating insulin signaling, fibrosis, and inflammation of the liver [57]. GPR39 is an orphan receptor because its endogenous ligand aside from zinc has not been identified unequivocally [58]. A specific aspect of intestinal zinc metabolism is the secretion of zinc ions from Paneth cells [59]. Here, ZnT2 has a role as an importer of zinc ions into cellular vesicles that secrete the zinc ions. The purpose of zinc secretion is thought to be for immune defense and/or controlling proliferation of stem cells [60,61]. Inside the enterocytes and other cells, additional zinc transporters function in zinc re-distribution and signaling pathways [62].

In addition to ZIP4, other examples of how the expression or mutations of specific zinc transporters in the gut affect overall health include ZnT2 and ZIP8, which is associated with myeloid cells [63,64]. Numerous polymorphisms in the dozen genes of MTs that serve as controlled and dynamic zinc buffers also affect cellular zinc homeostasis [65]. Zinc availability in the gut depends on the presence of chelating agents such as phytate in plant-based food. Since phytate lowers zinc uptake it has been called an antinutrient. The more phytate is in the diet, the more dietary zinc is needed [66].

Yet other zinc transporters such as ZIP2 route zinc across the membrane of endothelial cells into the portal vein. Once in the portal system, zinc is carried to the liver and later released into the hepatic vein to deliver it to other tissues through the systemic circulation [67]. In this way, the liver controls zinc concentrations in the blood and zinc distribution to other organs. Under normal physiological conditions, the zinc level in the body is tightly regulated through coordinated and combined mechanisms of absorption, transport, and secretion [68,69]. Zinc status in the body and zinc concentrations in the diet affect the efficacy of absorption and intestinal secretion of endogenous zinc [70]. Thus, the gut-liver axis is in fact bidirectional, delivering zinc to the liver through the portal vein and bringing zinc to the gut through several arteries and biliary and pancreatic secretions in a retrograde liver-gut axis. The secretion of 1–3 mg/day zinc into the intestine in this enterohepatic circulation is important as it is part of the control of systemic zinc through intestinal re-absorption.

Almost 95 % of body zinc is located intracellularly. The intracellular zinc concentrations are 10–20 times higher than the extracellular zinc concentrations and both compartments interact. How this gradient is achieved is not entirely clear in terms of what drives zinc transport through the plasma membrane energetically. The zinc transporters of the ZIP family are not coupled to ATP hydrolysis. Zinc in blood is mainly bound to albumin (60 %) and α₂-macroglobulin (20 %) with the remainder bound to other proteins such as transferrin. Yet, zinc remains accessible to membrane transporters that control the cellular zinc balance [68,70]. The liver has a major role in the synthesis of these blood proteins.

4

Role of zinc in intestinal barrier function

4.1

Functions of the intestinal barrier

In addition to the general functions of zinc for every intestinal cell, there are at least four specific areas where zinc is important for gut health: i) maintenance of barrier function, ii) expression of mucins, iii) homeostasis of immune cells, and iv) the composition of the gut microbiome.

Aside from the uptake of nutrients, the gastrointestinal tract has a critical role in protecting the host against pathogens and dangerous substances. A “leaky gut” brings bacteria and their products and other substances with toxic actions into the portal vein and can cause liver injury and affect liver regeneration. A robust system of physical, chemical and biological barriers affords this protection [71]. When the barrier is compromised antigens in the gut lumen can enter the host, trigger immunological and anti-inflammatory responses, and increase the host’s susceptibility to diseases of the gastrointestinal tract, e.g., chronic inflammatory disease [71]. Importantly, this inflammation also affects the development and progression of chronic liver disease. Thus, maintaining an intact intestinal barrier system and a healthy intestinal ecology of microorganisms is important for the health of the gut, the liver, and consequently the entire organism (Fig. 4).

Fig. 4.

The role of zinc in gut and liver disease. External factors (blue) and the gut microbiome (blue) are factors in gut disease. Importantly, gut disease (red) is a predisposing factor for liver disease (red). Zinc (Zn) is involved in all stages and, in addition, has roles in the immune system. Genetic predisposition, dietary zinc deficiency or other external factors that affect zinc uptake are also important in pathogenesis.

The intestinal barrier has multiple functions: acquiring nutrients, synthesis of metabolites, resorption of metabolites such as BA, vitamins, short-chain fatty acids, maintenance of both epithelial integrity and development and the innate and adaptive immunity, and thus immunological functions against pathogens [5,72]. Tight junctions and their specific proteins are critical for maintaining the intestinal epithelial barrier function. Environmental factors such as diet have a major role in regulating the gut epithelium and its barrier function [73]. Nutritional supplements, e.g., probiotics, vitamins (Vitamin D) and microminerals (zinc), have a positive influence on the integrity of the epithelial barrier and its permeability [74–76].

4.2

Zinc deficiency: impact on barrier functions

Zinc is involved in maintaining the structure and function of the membrane barrier and a healthy epithelium, thereby contributing to the host’s defense, which is particularly relevant in the intestine as it is continuously exposed to noxious agents and pathogens [77]. The role of zinc deficiency in compromising intestinal barrier functions was demonstrated succinctly in a mouse model of Shigella flexneri-induced diarrhea [78]. The observation of bacterial translocation, colonization and shedding and a significant perturbation of the intestinal permeability established zinc deficiency as a pathogenic factor for the development and extent of bacterial infection and ensuing inflammation.

Zinc deficiency can lead to a “leaky gut” through an increase of nitric oxide production, oxidative distress, resulting in an increased risk of bacterial infection and ensuing diarrhea [50]. It changes the integrity and function of epithelial cells and their gap junctions and tight junctions through reduced expression of proteins such as occludin and claudin [79]. Focal leaks increase intestinal permeability and thus leakiness, triggering an immune response [71,80]. Several independent investigations have shown that zinc deficiency, which can also be triggered by various diseases, is associated with a compromised barrier function [81]. Malnutrition [82], alcoholic liver disease [83], and chronic inflammatory gut disease can lead to a zinc deficiency state [77]. Also, in bacterial infections during dialysis the mucus barrier becomes more vulnerable [84]. Vice versa, zinc supplementation protects against intestinal dysfunction and leakiness and improves barrier function [85].

Chronic, excessive alcohol consumption interferes with the intestinal microbiome, affects the intestinal immune system, and can lead to intestinal inflammation and increased gut permeability [86]. In ALD patients, an increased intestinal permeability is associated with epithelial damage and histological changes in the intestinal mucosa. The latter includes villous atrophy, increases in lamina propria infiltrate and intraepithelial lymphocytes and changes in cellular functions, e.g., the brush border membrane and cellular enzymes. In healthy humans, acute binge drinking increases endotoxin and bacterial DNA concentrations in the blood [87]. Significantly increased blood endotoxin concentrations were measured in patients with different stages of alcoholic liver injury, including steatosis, hepatitis and cirrhosis, and in experimental animal models of ALD [88,89]. Zinc deficiency is observed under chronic alcohol abuse and correlates with increased intestinal barrier dysfunction and gut permeability and thus an increased endotoxemia in alcoholic liver damage [90].

Recent investigations have revealed molecular pathways for zinc’s functions in the intestinal immune response and its protective effects on the intestinal epithelial barrier [91,92]. In mouse intestinal organoids, HDAC (total histone deacetylase) and zinc uptake are reduced as a result of ZIP14 ablation [93]. ZIP14 gene deletion increases intestinal permeability (differential expression of claudin 1 and 2) and expression of interleukin-6 (IL-6) and IFN-γ, resulting in mild endotoxemia, intestinal dysbiosis, and compromised immunity through decreased expression of major histocompatibility complex II [94]. Zinc supplementation of the organoids reversed the effects on gene expression and demonstrates that zinc-dependent HDACs obtain zinc from ZIP14-mediated import and that intestinal integrity is at least partially controlled by epigenetic modifications.

When the influence of prior zinc administration on indomethacin-induced inflammation of the small intestine was investigated in a mouse model, the vehicle group (without zinc) showed destruction of the villi architecture, a shortening of the small intestine and a significant increase in both alkaline phosphatase (AP) and myeloperoxidase (MPO) activity as an indicator of inflammation [95]. Two days after indomethacin administration, the expression levels of the zinc transporters ZIP4 and ZIP14 were reduced in the small intestine. In the zinc-pretreated group, the inflammatory changes in the villi were much less pronounced, AP activity increased while MPO activity decreased significantly. Thus, zinc is effective in preventing NSAID (non-steroidal anti-inflammatory drug)-induced intestinal inflammation, which is a cause of reduced zinc absorption.

An important pathway for the health-promoting effects of zinc on the intestinal epithelial barrier function involves the aryl hydrocarbon receptor (AHR) [96,97]. The activation of AHR with plant-derived ligands present in our diet leads to down-stream signaling and gene expression of zinc importers of the ZIP family in intestinal epithelial cells, which in turn generate cellular zinc ion signals that control expression of tight junction proteins, thus decreasing leakiness and being beneficial in preventing inflammatory bowel disease (IBD).

4.3

Role of portal hypertension in intestinal permeability

While there are similarities in the molecular events in IBD and chronic liver diseases, portal hypertension is a typical feature of decompensated liver cirrhosis and a main driver of the systemic inflammation associated with liver cirrhosis, and it can occur in IBD [98]. It directly impacts the integrity of the intestinal mucosal barrier and impairs its function, leading to vascular congestion, intestinal wall edema, and widening and damage to the tight junctions [99,100]. Portal hypertension is thus a decisive factor for the increased intestinal permeability and for the translocation of bacteria and their products into the liver and the systemic circulation. The portal blood pressure correlates significantly with intestinal permeability, and placement of a transjugular, intrahepatic portosystemic shunt decreases portal pressure and intestinal permeability [101]. The significant improvement of intestinal permeability and bacterial translocation after treatment with non-selective beta-blockers in the patients further supports a role for portal hypertension in affecting intestinal barrier function [101,102]. A molecular mechanism involves nitric oxide, which has a key role in the haemodynamic changes associated with cirrhosis and portal hypertension [103], but can also increase intestinal permeability [98,102]. Changes in the intestinal mucosa and shortened and widened microvilli were observed in cirrhotic patients with portal hypertension ([101], [104–106]).

4.4

Role of mucins

The mucus layer has a role in metal coordination [107]. Mucins, glycoproteins produced by Goblet cells, have a role in capturing metal ions and thus keeping them in a soluble form for uptake when the metal ions transit from the highly acidic stomach lumen to the slightly alkaline lumen of the small intestine [108]. Zinc deficiency affects the expression of mucins, which also have roles in copper acquisition [107,109], intestinal mucus stability, and the integrity of this physical barrier. While a major function of secreted mucus is the protection of the epithelium from commensal and pathogenic bacteria, the glycoproteins also provide a nutritious milieu for bacteria in the colon.

4.5

Effects of zinc administration on the intestinal immune cells and inflammation

Zinc is also important for the homeostasis of intestinal immune cells, and anti-diarrheal and anti-inflammatory activities [73]. A significant improvement in the inflammatory condition was found in a mouse model of colitis with dietary zinc supplementation, using zinc oxide nanoparticles [110]. A reduction in both the disease activity index and histological lesions was observed. Remarkably, the maximal protective effect was observed with 160 ppm (parts per million) dietary zinc, which turned out to be better than with 400 or 1000 ppm, respectively, emphasizing the importance of optimal zinc doses. The effects are based on the anti-inflammatory effect of zinc via suppressing inflammatory cytokines (IL-1ß and IFN-γ-inducing IL-18) as well as re-balancing the T-cell subset Th1/Th2/Th17 [110].

By modulating intestinal barrier permeability and functions in Paneth cells, zinc is needed for regulating the host-microbiome relationship [111]. A constant battle between the host and the microbiota for zinc is waged in the gut as the microorganisms also need zinc for survival. It is a complex process and includes the biosynthesis of zinc-chelating agents, zincophores, and zinc ionophores. The microbes and the host have complex ways of controlling ecological niches by restricting metal ions or making them available to intoxicate microorganisms [112,113]. Zinc from diet influences directly intestinal microorganisms [114,115]. Too low as well as too high zinc concentrations can cause dysbiosis of the microflora and affect metabolism by affecting the synthesis of short-chain fatty acid and other metabolites [111]. Short-chain fatty acids such as bacterially produced butyrate are important for energy metabolism and the health of intestinal cells as well as for the epithelium via suppressing inflammation [116].

An important consequence of microbial dysbiosis under zinc deficiency is the incidence of diarrhea, which itself is a cause for losing zinc. Diarrhea reduces the absorption of zinc because of rapid intestinal transit and destruction of the absorptive villous mucosa. Zinc supplementation decreases the duration and severity of diarrhea. Zinc has bactericidal properties and therefore is effective in infectious diarrhea [117–119]. A zinc deficiency reduces absorption of water and electrolytes as well as clearance of aetiologic pathogens, thus prolonging the resolution of diarrheal episodes [120]. A recent review addresses how diarrhea with an underlying zinc deficiency affects different organs [121]. Individuals with zinc deficiency have reduced lymphocyte proliferation in response to the exposure of mitogens and other changes, and zinc supplementation reverses these deficits [122].

An important observation that links inflammatory diseases of the gut and liver diseases is that at least 5 % of patients with Crohn’s disease and ulcerative colitis develop liver disease [123,124].

4.6

Interactions between primary sclerosing cholangitis and inflammatory bowel disease

Primary sclerosing cholangitis (PSC) is a rare, chronic progressive cholestatic liver autoimmune disease of unknown aetiology characterized by intrahepatic and extrahepatic chronic inflammation and progressive fibrosis of the biliary tree [123]. It is highly heterogeneous, generally leads to end-stage liver cirrhosis, and is a risk factor for hepatobiliary and colorectal neoplasms [124]. The relationship between PSC and IBD provides an opportunity to emphasize the role of the gut-liver axis not only in physiology but also in pathology. The liver produces various biological mediators, including BA, which enter the intestine via the bile ducts. In the intestine, the microbiota metabolize these BA and other nutrients, and this mixture then returns to the liver via the portal vein. Microbiome dysbiosis resulting from various intestinal diseases, acute or chronic inflammation, production of endotoxins, toxic secondary BA such as lithocholic acid and other metabolites can disrupt the intestinal barrier and have repercussions for the health of the liver [8,18,123].

Up to 80 % of PSC patients suffer from concurrent IBD, especially ulcerative colitis [18,123,125,126]. Conversely, only 2–10 % of patients with IBD suffer from PSC [124,126–128]. Several pathophysiological processes have been suggested as a link between the gut and the liver in PSC. There are currently four main hypotheses regarding a perturbed gut-liver axis in PSC: (i) a leaky gut, (ii) microbiome dysbiosis, (iii) altered bile acid signaling, and (iv) aberrant lymphocyte trafficking. As described above, chronic intestinal inflammation in IBD leads to damage to the intestinal barriers such that bacteria and their products, e.g., endotoxins, toxic BAs and short-chain fatty acids, enter the liver and cause inflammation and fibrosis to the point of cirrhosis as well as progressive scarring of the bile ducts [129,130]. Since there is currently no effective therapy that can change the natural course of PSC, liver transplantation remains the last treatment option. If PSC occurs at the same time as IBD, the risk of developing colorectal neoplasia also increases [123].

Malabsorption disorders are closely associated with micronutrient deficiencies. In IBD, trace element deficiencies pose a clinical burden from disease onset throughout its course and contribute to morbidity and poor quality of life [131]. Among a total of 1677 patients with Crohn's disease and 806 patients with ulcerative colitis, almost half of the patients have a zinc deficiency, and the main cause was seen in inadequate food intake and in the malabsorption in IBD [131]. The reduced zinc levels contribute to increased intestinal inflammation and thus dysfunction, compromised immunity, and ultimately a further increase in pro-inflammatory cytokines [131]. Zinc deficiency induces membrane barrier damage [132]. Based on these observations, zinc supplementation is recommended to stabilize the intestinal barrier function. From the experience of one of us (KG), the administration of glucocorticoids in the acute stage of the disease also contributes to zinc deficiency [133]. When the effect of zinc supplementation on cholestatic liver disease was investigated in a mouse model, pathological liver changes decreased [134]. The improvement was not due to an influence on the BA in the liver but a result of modulation of the intestinal microbiota, specifically an interaction of zinc with Blautia producta-produced p-coumaric acid, which ameliorates cholestatic liver disease by reducing hepatocyte pyroptosis induced by BA. While these investigations prove the positive effect of zinc administration on cholestatic liver disease, questions remain as to the dosage and duration of zinc medication.

5

The gut as an immunological organ

5.1

The microbiome

The gut microbiome is a community of trillions of microorganisms that inhabit our gastrointestinal tract and interact with dietary compounds bidirectionally, being impacted by their composition as well as influencing their metabolism and bioavailability [135,136]. Micronutrients affect the composition and diversity of the gut microbiota and thus their effects on the host organism with either positive or negative outcomes [135]. The multifaceted bionetwork of bacteria, archaea, viruses, fungi, and protozoa exceeds significantly the number of human “self” cells that passed the germline [137]. The human intestinal microbiota holds almost 100 times more genetic material than the human genome [138]. This massive microbial community is composed of the five phyla of bacteria: Bacteroides, Actinobacteria, Firmicutes, Verrucomicrobiota, and Proteomicrobiota [137]. In the context of host physiology, the commensal microbiome has a vital role in numerous processes including fiber breakdown, vitamin production, e.g., biotin, folate, and vitamin K, xenobiotic metabolism, and immune system function and maturation [137]. Importantly, and with relevance to the discussion here, the microbiome itself needs zinc and has a role in determining the availability of this and other micronutrient metal ions for the host. Gut microbes also have essential roles in the synthesis and metabolism of BA [139].

As an endocrine organ, the gut is involved in energy homeostasis and immunity of the host [140]. While the microbiome changes significantly during the life course, it is relatively stable in healthy adult individuals. However, in chronic metabolic diseases, which are often accompanied by subluminal inflammatory processes, e.g., diabetes mellitus, obesity, significant changes in the composition of the microbiome have been observed. With relevance to liver disease, the microbiome of cirrhotic patients is characterized by an overgrowth of potentially pathogenic germs and a reduction of autochthonous, non-pathogenic bacteria [8]. Moreover, a fungal microbiome (mycobiome) has been identified in cancer [141,142].

The zinc status of an individual affects the microbiome. When groups of zinc deficient and zinc adequate school children were examined regarding their serum zinc levels, anthropometric measurements, metagenomes and metabolomes, and a large number of stool analyses of the composition and function of the intestinal flora, the two groups had significant differences in the microbial composition [143]. The effect of zinc deficiency on the intestinal flora affects metabolic functions. For example, mucin-reducing bacteria such as Phocaucola vulgaris were more common in those school children with zinc deficient sera. Higher levels of taurocholic acid and saccharin were associated with damage to the intestinal barrier and a pro-inflammatory condition, which ultimately led to an increase in malabsorption and development of zinc deficiency. The investigation underscores impressively the importance of zinc for the composition of the intestinal microbiota and associated metabolic processes in the host. The effects of zinc deficiency on the gut microbiome were corroborated in another investigation that demonstrated changes in bacterial taxa with functions in multiple metabolic processes: “The changes in α-diversity and short-chain fatty acid production, however, were inconsistent. In contrast, high dietary zinc/zinc overload generally led to either unchanged or decreased α-diversity, decreased short-chain fatty acids, and an increase in bacterial gene expression for proteins involved in metal and antibiotic resistance [144].”

5.2

Metabolic endotoxemia

Metabolic endotoxemia is defined as a diet-induced 2- to 3-fold increase in plasma LPS associated with low-grade inflammation, ultimately leading to the development of cardiometabolic diseases [145,146]. It has been proposed as a common denominator of the strong linkages among obesity, inflammation, impaired glucose tolerance, and the development of type 2 diabetes [145,147,148]. Structural changes to the intestinal epithelium and a leaky gut allow LPS to enter the bloodstream. LPS is an important pathogen-associated molecular patterns (PAMPs) factor in gram-negative bacteria, of which the smallest amounts in the blood due to a bacterial infection are sufficient to trigger an inflammatory response [149]. LPS activates Toll-like receptor-4 (TLR4), leading to the production of numerous pro-inflammatory cytokines and associated low-grade systemic inflammation.

Dietary factors and eating patterns play a critical role in the modulation of the gut microbiota and thus metabolic endotoxemia [150]. Changes to a high fat/high sugar diet from a low fat/polysaccharide-rich plant diet in mice [151] as well as from a high fat/low fiber diet to a low fat/high fiber diet in humans [152] markedly altered the microbiome in the gut within a day. Remarkably, the consumption of a Western diet (high fat, high sugar) for one-month increased endotoxemia in healthy individuals when compared to the so-called Prudent diet (low fat, high levels of fruits, vegetables, whole grain, poultry and fish) [153,154].

Consistent with animal models, several investigations have reported elevated levels of blood endotoxins in adult patients with simple steatosis, hepatitis and NASH [150]. Intestinal permeability was increased in children with steatohepatitis compared to those with only steatosis [155]. The high fat meals [155] or drinks [157] resulted in low grade endotoxemia in healthy humans over time, possibly by mechanisms involving increased intestinal LPS absorption through incorporation into chylomicrons [150,158]. Endotoxins increased in NAFLD adolescents after consumption of fructose-containing beverages, underscoring the role of dietary factors in gut barrier integrity [159]. However, the levels of LPS in blood after consumption of the above diet are 10–15 times lower compared to those seen in animals or humans with sepsis [145]. Thus, metabolic endotoxemia causes a state of low-grade inflammation, which is a pathogenic factor for a range of chronic conditions including type 2 diabetes mellitus, NAFLD, chronic kidney diseases, and atherosclerosis [160]. A positive correlation also exists between the quantity of alcohol consumed and serum LPS concentrations [161]. Larger amounts of alcohol can increase intestinal permeability and thus LPS levels in the systemic circulation and are also associated with changes in the intestinal microbiota that increase LPS levels [86].

With the detection of elevated LPS levels in various other conditions in recent years, the initially controversial attitude towards the concept of metabolic endotoxemia has given way to its general acceptance [162]. Increased LPS levels were observed not only in ALD [83,163,164], but also in NAFLD [140], obesity [165], type 2 diabetes mellitus [166], pancreatitis [167], and neurodegenerative diseases [168]. In addition to the liver, LPS also trigger low-grade inflammation in other organs [169].

Among the mechanisms discussed for the translocation of LPS in metabolic diseases are (i) a leaky gut [146]; (ii) hyperglycemia in diabetes mellitus [170]; and (iii) high fat meals that affect either chylomicron-associated transfer [158] or internalization by intestinal microfold cells [171].

Investigations on patients with severe chronic alcoholic liver injury revealed significantly decreased serum/plasma zinc levels in the transition from the subclinical stage to fully developed ALD, i.e., alcoholic fatty liver disease and mild alcoholic hepatitis [172]. A low serum/plasma zinc level is considered an important factor in the gut-liver axis to accelerate intestinal dysfunction, liver inflammation, hepatocyte damage and necrosis in the early stages of ALD. Therefore, recording the blood zinc level together with proinflammatory cytokines and hepatocyte markers is recommended as a diagnostic tool in the early stages of ALD [172].

In summary, the evidence suggests starting points for the therapy of metabolic diseases, including chronic liver diseases [146] through changes in eating behavior (composition and amount of food and timing of food intake), the use of probiotics, and essential micronutrients such as zinc [146,162].

6

Zinc metabolism in the liver

The liver has a central role in the metabolism of zinc and other metal micronutrients [173]. It has a relatively fast (turnover two hours) and a slower (turnover 20 hours) exchanging zinc pool, owing to the actions of multiple zinc homeostatic proteins such as MTs and ZIP and ZnT transporters that are regulated by hormones such as insulin, glucagon, and glucocorticoids [1,174–176] and by cytokines such as IL-6. Depending on the metabolic situation, these effectors trigger transient changes in zinc concentrations. For example, elevated plasma glucose after a meal correlates with decreased plasma zinc [177], which is thought to be mediated by ZIP14 transporting zinc into the liver [178].

The crosstalk between the liver and the pancreas regarding zinc and metabolic hormones is also critical for zinc metabolism. Insulin is stored in the β-cells of the pancreas as a crystalline hexamer that is stabilized by two zinc ions and one calcium ion. The dilution of zinc co-secreted with insulin from pancreatic beta-cells in the blood and complexation by other proteins such as albumin promotes the formation of an active insulin monomer that is also the form that is cleared by the liver [179,180]. In this way, plasma zinc levels and speciation are also thought to be affected by hepatic insulin clearance [179]. Experiments involving subcutaneous injections suggest that only the insulin monomer can pass through the blood vessel wall [181,182]. In target tissues of insulin such as the liver, zinc is an inhibitor of protein tyrosine phosphatase 1B, which regulates the tyrosine kinase activity of the insulin receptor and accounts of the insulin-mimetic properties of zinc [183]. Vice versa, zinc deficiency interferes with this control and thus is a cause of insulin resistance. Furthermore, the zinc co-secreted with insulin has an autocrine action on β-cells and a paracrine action on glucagon-secreting α-cells [184].

Systemic zinc deficiency influences different hepatic functions and, because of the central role of the liver in zinc metabolism, impacts metabolic processes in other organs [1]. Changes in cellular zinc availability directly affect gene expression [185]. An increased dietary intake of zinc triggers MT synthesis [174]. MTs buffer the cellular zinc ion concentrations dynamically and in this way control many zinc-dependent cellular functions [65]. Interactions also have been identified among zinc, copper, iron, and IL-6, one of the most important pro-inflammatory cytokines regulating the gene expression of acute phase proteins. The uptake of zinc in the liver in the acute phase reaction is linked to the regulation of gluconeogenesis, the control of reactive species, e.g., nitrogen monoxide, and the control of microbial growth through the nutritional immunity established by removing zinc and iron from blood while increasing copper. The effects of zinc in the acute phase response are mediated by IL-6. For example, IL-6 upregulates ZIP14 in hepatocytes and induces hypozincemia (decreased plasma zinc levels) in the acute phase response [31], an aspect of nutritional immunity. ZIP14 also transports manganese and non-transferrin-bound iron and influences serum iron (hypoferritinemia) in the context of inflammatory processes [186]. Hepatic ZIP14 is also responsible for uptake of zinc during liver regeneration [178] and has a critical role in the development of tumors, e.g., in hepatocellular carcinoma, HCC [187,188].

Albumin is the main carrier of zinc in the blood, and hence critical for its systemic distribution. It binds serum zinc with a molar ratio of 30:1 that is kept within narrow limits, and changes in one are always accompanied by changes in the other. Albumin is a negative acute phase protein. A decrease in albumin concentrations, for example, in inflammatory situations, accompanies a decrease in zinc concentrations, which results in a change in protein metabolism and a reduction in urinary nitrogen excretion, and reduced concentrations of prealbumin and retinol-binding protein in serum [27]. With this interaction between zinc and albumin, reduced protein intake is also associated with reduced zinc intake [27,69]. Another important aspect is the competitive binding of zinc and certain fatty acids to albumin. It establishes a metabolic relationship between zinc and lipid metabolism [189]. Increased concentrations of certain fatty acids as observed in obesogenic diets and in metabolic syndrome displace zinc from albumin and thereby affect zinc buffering in blood, and presumably its availability and use.

7

The liver as an immunological organ

The liver is also a critical immunological organ with unique properties that set it apart from other lymphoid tissues. Its strategic location, receiving blood from the systemic circulation and the gut via the portal vein, positions it as a frontline defender against pathogens and toxins [190].

The liver contains different immune cells, each type contributing to its complex immunological functions: (i) Kupffer cells, specialized macrophages that are involved in phagocytosis of pathogens and cellular debris, antigen presentation, cytokine production and modulation of T cell responses; (ii) liver sinusoidal endothelial cells (LSECs), which serve as antigen-presenting cells, express scavenger receptors for pathogen recognition and contribute to immune tolerance through antigen cross-presentation; (iii) hepatic stellate cells involved in antigen presentation and T cell activation and suppression; (iv) hepatocytes, which are producer of APP, secrete complement components and express pattern recognition receptors; (v) Natural Killer (NK) cells, which are enriched in the liver compared to peripheral blood and are critical for early defense against viral infections and tumors, and produce cytokines such as IFN-γ and mediate cytotoxicity against infected or transformed cells; (vi)Natural Killer T (NKT) cells, which recognize lipid antigens presented by CD1d molecules and bridge innate and adaptive immunity; (vii) conventional T Cells, both CD4+ and CD8+ T cells, which often display an activated or memory phenotype and can contribute to both protective immunity and liver injury; (viii) regulatory T Cells (Tregs), which play a crucial role in maintaining liver immune tolerance, suppress excessive immune responses and are important for preventing autoimmune liver diseases [190–192].

Immunological functions of the liver include pathogen detection and clearance, antigen presentation and T cell priming, enabling of immune tolerance, systemic immune regulation by production of acute phase proteins during inflammation, secretion of complement components and clearance of activated immune complexes from the circulation as well as immunosurveillance provided by NK cells, NKT cells and Kupffer cells.

7.1

Acute phase response and acute phase proteins

The acute phase reaction, i.e., expression of APP such as C-reactive protein (CRP), ferritin, caeruloplasmin, fibrinogen, plasminogen, complement factors (C3, C4), procalcitonin together with the innate immune system is one of the decisive responses against damaging influences [193–195].

The liver, which is constantly challenged by various pathogens, allergens and other damaging influences through the blood and splanchnic systems, has a central role as APP are synthesized in the hepatocytes where liver macrophages (Kupffer cells) have a key role in the regulation of the expression [193,196,197].

During an inflammatory response, a typical change of serum proteins occurs in addition to the expression of APP, which in combination with clinical symptoms (fever, chills, exhaustion) are called the acute phase response [198,199].

CRP is an acute phase reactant that increases in response to various inflammatory stimuli, such as infections, injuries and autoimmune disease [200]. CRP activates macrophages to secrete tissue factor that has a coagulant function [201]. As an indicator of inflammation that increases with age, CRP can contribute to the development of NAFLD in aging by mediating insulin signaling, mitochondrial metabolism, and NF-κB signaling. In growth and development, metabolism, and apoptosis, CRP is an important link among chronic inflammation, aging and NAFLD [200]. Hepatocytes are the main parenchymal cells in the liver, making up about 80 % of total liver cells [202]. Hepatocytes participate in injury as antigen-presenting cells and activate innate immunity through secreting immune molecules such as CRP [203,204]. The production of CRP in hepatocytes is mainly regulated by cytokines, especially IL-6 [205]. Acute phase proteins, e.g., A1AT (alpha-1 antitrypsin) or CRP, serve as anti-inflammatory proteins under certain conditions [206–208].

The liver also has a critical role in the regulation of the trace elements during the acute phase response. At the onset of systemic injury and infection, the acute phase response is activated, leading to the mobilization of zinc and iron from the blood compartment into the liver and the release of copper, which is also part of the nutritional immunity, i.e., starving microorganisms by depriving them of metal ions that are essential for their growth. The alterations in zinc metabolism are critical in guiding the initial host response that influences both innate and adaptive immune function [209]. If the host is zinc-sufficient, the chance of recovery improves, whereas zinc deficiency increases morbidity and mortality. The cellular consequences of zinc deficiency are manifested through oxidative stress, increase of inflammation, and in extreme situations premature cell death [210]. Glucocorticoids as stress mediators also influence this process [211].

The battle for micronutrients between the vertebratehost and pathogenic microorganisms is a fundamental aspect of a "first line of defense” against invasive pathogens and is called "nutritional immunity" [212–214]. During infections, pathogens need Zn²⁺ in competition with the host to colonize and cause disease [215]. Immediately after penetration of the pathogen, the organism reacts by reducing serum zinc levels [212,216].

7.2

Chronic inflammation in liver cirrhosis

Inflammation is an essential process of innate immunity to protect the host against invasion of pathogenic challenges or damage to tissue [217]. Mononuclear phagocytes, especially monocytes and macrophages, have an essential role in initiating, regulating, and resolving the immune reactions by phagocytosis, production of cytokines or reactive species, and activation of adaptive immunity [217]. Normally, inflammation is resolved if the pathogen is inactivated, or the damaging influences cease, and the tissue defect is repaired. The development of chronic inflammation is a decisive factor in the development of chronic disease, such as liver cirrhosis [1,218].

Infections increase the mortality in patients with liver cirrhosis significantly and thus are a cause for poor prognosis. 30 % of patients with liver cirrhosis die within a month with an accompanying infection, and another 30 % within a year [219]. The most common infections are spontaneous bacterial peritonitis (SBP), urinary tract infections, pneumonia, and skin infections [7,220,221].

The abnormal translocation of bacteria and pathogen-associated molecular patterns (PAMPs) into the portal and then the systemic circulation is a major mechanism of developing systemic inflammation in liver cirrhosis. DAMPs as well as PAMPs bind to pattern recognition receptors (PRR) and trigger an activation of hepatic and extrahepatic immune cells. In this way, moderate systemic inflammation develops, in addition to the inflamed gut and liver, already at the stage of compensated liver cirrhosis [222]. In decompensated liver cirrhosis, systemic inflammation increases further.

These interactions emphasize the importance of the liver-gut axis and the microbiome in chronic disease of the liver, specifically liver cirrhosis [223–225].

The liver's role as an immunological organ is complex and multifaceted. Its unique anatomy, diverse cellular components, and specialized functions contribute to both local and systemic immune regulation. Understanding the liver's immunological properties is crucial for developing new therapeutic approaches to liver diseases and harnessing its potential in treating systemic immune disorders. As research in this field continues to evolve, the liver's importance in immunology becomes increasingly apparent, opening new avenues for medical interventions and deepening our understanding of the intricate relationship between metabolism and immunity.

8

Zinc deficiency

8.1

General considerations

Zinc deficiency [226], the systemic inadequacy of zinc required to support its many fundamental cellular functions, is remarkably prevalent. Approximately 17 % of the global population are at risk of becoming zinc deficient, particularly in low-to-middle income countries. Zinc deficiency is an important public health issue. In children, it is a significant factor for mortality through diarrhea and infections [1,227–229]. The determination of zinc concentrations in serum or plasma with defined trace metal free collection systems and at defined times of phlebotomy is often considered as the most appropriate measure of zinc deficiency in clinical practice and for determining zinc status in populations. In official policy documents, the term „zinc deficiency" describes a reduction in serum zinc levels from the normal range of 78.5–117.7 µg/dL (12–18 µM) in serum/plasma to below 70 µg/dL in women and 74 µg/dL in men with corresponding clinical symptoms [230].

Plasma/serum zinc concentrations are subject to influences such as diurnal fluctuations [231,232], the nocturnal fasting period, the extent and timing of the last meal [233] and inflammation [234]. There are numerous other potential functional biomarkers for assessing zinc status, such as fatty acid desaturation, DNA damage, and zinc transporter gene expression, but none of them have been validated or implemented clinically [231,235]. Data from animal models suggest that changes in the intestinal microbiota could be used to assess zinc status [231]. Before any of these new functional parameters, which appear to be suitable as experimental markers for zinc status, can be widely applied, they must be tested in extensive clinical intervention studies to determine whether they are markers of a general zinc deficiency or indicate a specific functional deficit, are feasible in practice, reproducible, and safe. Based on many years of our own clinical experience, the determination of zinc concentrations in serum/plasma under standardized conditions is a reliable parameter for assessing adequate zinc status [236–238].

Symptoms of moderate to more severe zinc deficiency include mental lethargy, poor appetite, altered smell and taste, loss of body hair, delayed wound healing, testicular atrophy, immune dysfunction, and diminished drug elimination capacity [226]. Primary zinc deficiency is caused by insufficient intake, malabsorption, or in rare cases, genetic predisposition. Conditioned zinc deficiency is dependent on other factors that limit the ability to take up or utilize zinc. It is observed in chronic disorders of the heart, liver, lung, and pancreas [226,239,240]. Many, if not all, of these disorders are associated with systemic inflammation and oxidative stress, e.g., diabetes mellitus, obesity, and autoimmune diseases such as rheumatoid arthritis. Zinc deficiency becomes more prevalent with age, which is thought to be linked to an increase in inflammatory processes and senescence (“inflammaging”), and is manifested in reduced immune function, and thus a factor in the increased prevalence of diseases in the elderly [241].

Japan's practical guidelines [242] once again impressively underline that zinc deficiency is not only a "laboratory diagnosis" but also includes anamnestic data and clinical findings. As already mentioned above, hair loss, weight loss, reduced tactile sensation or increased infections, among other symptoms, indicate a possible zinc deficiency. Diseases that are often associated with zinc deficiency include diabetes mellitus, cirrhosis of the liver, chronic kidney disease, and many others.

When the effects of zinc deficiency on the rat small intestine immune barrier - B cells, plasma cells, effector T cells and innate lymphoid cells – were investigated, a reduction in intestinal immunoglobulin IgA, an integral component of the mucosal barrier, was noted [243]. Zinc deficiency induces an imbalance of the gut microbiota, which leads to a local invasion of Gram-negative bacteria and subsequent spread via the portal vein into the liver, triggering an immunological defense reaction with hepatic increases in TLR4+ (CD284), CD68 monocytes, macrophages and neutrophils. The administration of IL-4 and zinc showed a positive effect on GALT (gut-associated lymphoid tissue) function, such as reduction of LPS levels in the portal vein and bacterial translocation to the liver. Thus, the administration of either IL-4 or zinc in zinc deficiency can directly suppress the inflammatory response [243].

8.2

Zinc deficiency and liver disease

Patients with liver disease frequently present with classical symptoms of zinc deficiency, particularly patients with liver cirrhosis [1,244–246]. The deficiency is caused by a variety of factors such as inadequate zinc intake, changes in protein and amino acid metabolism (including hypoalbuminemia), diminished hepatic extraction, portosystemic shunts, alcohol-induced impaired absorption, and effects of cytokines, mainly IL-6, and bacterial endotoxins.

Most patients with decompensated liver cirrhosis are malnourished and the malnutrition is associated with low serum zinc [247]. They show albumin deficiency with a consecutive zinc deficiency as albumin is the main transport protein for zinc in the blood. Therefore, depending on the extent of malnutrition, targeted and controlled oral nutrition or infusion must be given. In the case of pronounced ascites, albumin administration or the administration of branched-chain amino acids (leucine, isoleucine, valine) (BCAA) is also necessary. Likewise, if there is a proven deficiency of zinc, magnesium, or iron, targeted substitution must be considered.

One in three patients with liver disease has depression. Inflammatory processes are the link in this association. The systemic inflammatory milieu in many patients with liver disease is similar to that of patients with depression. The composition of the microbiome and an increased intestinal permeability seem to be a significant factor. Depression as well as liver disease, stress, alcohol, and the aging processes lead to a disturbed permeability of the intestinal mucosa, so that bacteria and their products, endotoxins, enter the systemic circulation and in this way trigger and maintain both liver diseases and depression [248]. Disorders in the balance of trace elements are important factors in initiating and maintaining these inflammatory processes. A recent review article recapitulates the importance of zinc in protecting the liver and the relevance of zinc in liver disease and its treatment [249].

Cirrhotic patients with ascites are catabolic and present with a massive reduction of muscle mass and function (sarcopenia). Diuretic therapy in these patients results not only in increased renal zinc excretion but also in reduced serum albumin and thus reduced zinc bound to albumin [250]. Hence, zinc must be substituted after diuretic therapy. From the age of 50 onward, many people experience a decrease in muscle mass of 1 % per year because of qualitative and quantitative changes in muscle fibers [251,253]. Frequently, zinc deficiency can be detected in the blood of sarcopenic patients. Sarcopenia is becoming increasingly important for scrutiny and treatment in chronic liver disease patients. Nearly 70 % of patients with decompensated liver cirrhosis have visible muscle loss with thin arms and an increased waist circumference due to ascites. Muscle loss is often already seen in NASH combined with obesity. Factors that induce sarcopenia in liver cirrhosis include hyperammonia, lack of branched-chain amino acids (Val, Leu, Ile) and testosterone, and increased concentrations of myostatin [253]. Cirrhotic patients with zinc deficiency have a significantly higher rate of developing sarcopenia [253]. Based on the results of a multivariate analysis of 372 cirrhotic patients, zinc deficiency occurred as an independent predictive factor for the development of sarcopenia in chronic liver disease [252,254]. Old age is a risk factor for liver disease and treatment options are unsatisfactory [255]. Because of the increased tendency to become zinc deficient at older age and the incidence of inflammaging, zinc should be considered in the treatment options in age-related liver disease.

8.3

Zinc deficiency and oxidative stress - is zinc an antioxidant?

Like calcium and magnesium, but unlike iron, copper, or manganese, zinc is redox-inert in biology and thus cannot be an antioxidant as is often claimed, though it is indirectly involved in redox metabolism and is necessary for the defense against a slew of “offenders.” One major role of zinc is in cytosolic and extracellular superoxide dismutase in a structural site next to the catalytic copper ion, but also in other enzymes involved in redox biology, namely nitric oxide metabolism, glutathione metabolism, and in MTs [256]. Notably, in addition to inducing proteins involved in the antioxidant response through the zinc-sensing transcription factor MTF-1 (metal regulatory transcription factor-1), zinc affects signaling to the transcription factor Nrf2 (nuclear factor erythroid-2 related factor 2), which controls the expression of yet other proteins involved in the antioxidant response [257]. The effects of zinc deficiency in eliciting oxidative stress indeed demonstrate that an inadequate amount of zinc is a pro-oxidant condition that affects many processes. Functions can be restored when zinc is supplemented, thus connecting the phenomenology observed in zinc deficiency with specific molecular processes and pathways.

9

Zinc supplementation in liver cirrhosis

9.1

General considerations

Based on our current understanding of the importance of zinc for biological processes and zinc deficiency as a pathogenetic factor in various disorders, the manifestation of typical zinc deficiency symptoms should trigger a determination of zinc in patients’ blood. In patients with chronic liver disease, independent of its genesis, especially in decompensated liver cirrhosis or a longer history of type 2 diabetes mellitus, monitoring of zinc levels should be instituted prior to the appearance of overt zinc deficiency symptoms, and zinc supplementation initiated when zinc levels in blood are reduced (< 11.00 µM or < 66 µg/dL) with or without symptoms. The maximum or optimal doses for zinc supplementation are currently not clearly defined but are relevant for long-term administration [1]. For instance, dosages of 100 mg or more “elemental” zinc per day can cause severe immunological damage [258,259]. Daily doses of >45 mg elemental zinc have adverse effects on immune cells with inhibition of DNA synthesis and cytokine production [260]. Oral zinc at <45 mg/day is not considered “outright toxic” [261].

However, the outcome of zinc supplementation in patients with liver cirrhosis is inconclusive. Recent meta-analyses revealed only marginal benefits of zinc on the clinical course of cirrhosis [262,263]. These conclusions are contrasted by many individual investigations of zinc supplementation that found a beneficial effect on metabolic disorders caused by zinc deficiency [264]. Reductions in ammonia levels, improvement of protein synthesis, decrease in insulin resistance, stimulation of liver regeneration [2,265] and several hepatoprotective effects were observed [264]. Improvement of insulin resistance and oxidative stress were reported with a 3-months zinc supplementation (30 mg elemental Zn) in obese patients with NAFLD [266]. Lipid profiles and body weight were not affected, however. The combination of zinc acetate and rifaximin had a preventive effect on ALD-related fibrosis in a mouse model [90]. This antifibrotic effect is attributed to an influence on multiple regulatory functions that maintain intestinal barrier integrity and reduce hepatic LPS exposure and lead to Kupffer cell expansion by inhibiting the TLR4 signaling pathway.

Zinc supplementation may counteract the increased gut permeability and the associated bacterial spillover into the systemic circulation in liver cirrhosis [267,268]. Zinc deficiency is thought to be associated with dysbiosis in patients with chronic liver disease [2] and involves a leaky gut in a mouse model of alcohol-induced steatohepatitis [83]. Vice versa, the administration of zinc sulfate preserves intestinal barrier function and improves endotoxemia in alcohol-induced steatohepatitis in rats [269]. Dietary zinc supplementation effectively corrects alcohol-mediated zinc deficiency and associated inflammation [269]. The protective effect of zinc against alcoholic endotoxemia is attributed to reduced luminal endotoxin loads, enhanced epithelial barrier function, and accelerated acetaldehyde clearance [269].

9.2

Hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that occurs in patients with acute or chronic liver disease and illustrates the role of the gut-liver-brain axis. Symptoms are compromised cognition and fine-motor movement at varying severity. Hyperammonemia and peripheral inflammation act synergistically to induce HE [270]. The former is sufficient to induce systemic inflammation, neuroinflammation, and neurological impairment [270]. Ammonia induces swelling of astrocytes, which disrupts neuronal energy production. Conversely, systemic inflammation induced by LPS treatment impairs hepatic ammonia detoxification by glutamine synthesis in rats [270] (Fig. 5).

Fig. 5.

Hepatic encephalopathy. Liver cirrhosis can lead to portosystemic shunt. A leaky gut, lack of metabolism of ammonia to urea in the liver, and reduced consumption of ammonia in muscle cause hyperammonemia, which together with systemic inflammation leads to neurological impairments. Zinc participates at several junctures as discussed in the main text. (Liver: Creator: eranicle | Credit: Getty Images. Copyright: eranicle; Brain: Creator: 3dMediSphere | Credit: Getty Images; intestine: Azat Valeev, Vecteezy, no attribution required. Copyright: Eraxion; for muscle: Vecteezy | License details Creator: Julee Ashmead | Credit: Vecteezy) Copyright: Julee Ashmead.

Compromised zinc status affects nitrogen metabolism by reducing the activity of urea cycle enzymes in the liver and glutamine synthesis in muscle [271–274]. It has been suggested that zinc deficiency has a role in the pathogenesis of HE as serum concentrations are reduced in patients and correlate inversely with blood ammonia concentrations [275]. The efficacy of zinc supplementation in HE is controversial. Positive effects [268,276–278] are contrasted by other investigations where no effect was observed [279]. A patient with severe recurrent HE and zinc deficiency improved with long-term zinc supplementation, concomitant with a decrease of blood ammonia levels [278]. Zinc supplementation has a beneficial effect on muscle cramps that occur frequently in patients with liver cirrhosis [280]. Positive effects of zinc supplementation in HE and on the overall condition of patients with cirrhosis were reported when zinc deficiency was confirmed [281,282]. A combination of zinc with two agents with different pharmacological properties appears to enhance efficacy in a way that is not achieved by either agent alone. Thus, the combination of zinc and BCAA or zinc and lactulose have an additive effect [283]. When groups with zinc supplementation were compared with both placebo and standard glucose therapy, there was significant improvement in the number connection test (NCT) but no effect on HE recurrence was observed [284]. The combination of zinc supplementation and lactulose over three to six months may improve the NCT in cirrhotic patients with low grade HE compared with lactulose only [285].

9.3

Branched chain amino acids and zinc in liver cirrhosis

The decrease in the capacity to synthesize albumin is a useful test for prognosis in liver cirrhosis. A major cause for the decreased albumin synthesis in liver cirrhosis is BCAA deficiency [249]. Aside from being necessary for protein structure, BCAA are involved in various biological processes, such as the stimulation of albumin and glycogen synthesis, improvement of insulin resistance, inhibition of reactive oxygen species (ROS) production and hepatocyte apoptosis, and liver regeneration [286]. The catabolic state of cirrhosis is characterized by an imbalance of plasma amino acids: a decrease of BCAA and an increase of aromatic amino acids (Tyr, Phe, Trp) [275,287]. In addition to changes in amino acid availability, protein catabolism leading to accelerated muscle breakdown and impaired ammonia removal in the liver and in muscle, patients with liver cirrhosis suffer from defects in glucose and fat metabolism with increased energy expenditure, often developing a hypercatabolic state [286,287].

The supplementation with BCAAs alone or in combination with zinc may contribute to an improvement of hypoalbuminemia and ascites through an increase in the supply of amino acids for protein synthesis and stimulation of protein synthesis [286,288,289]. Besides its oncotic effects, albumin has pleiotropic non-oncotic activities that include transport of endogenous and exogenous substances such as fatty acids and microminerals, antioxidants, immunomodulatory and detoxification functions, and stabilizing effects on the endothelium [264].

The efficacy of BCAA-rich supplements is based on two mechanisms. First, administration of BCAA is necessary to support protein synthesis. Second, through their nutrient-sensing functions and stimulation of intracellular signal transduction, BCAA increase mRNA expression and promote protein synthesis [290–292]. Specifically, continuous supplementation with BCAA induces phosphorylation of the ribosomal protein S6, which is a regulator of translation and a down-stream target of PI3K/AKT signaling, in livers of rats with chronic liver disease [292]. Thus, both zinc and BCAA are necessary for protein synthesis, increase of serum albumin and lowering the amount of ascites [268]. Zinc supplementation also significantly improves other nutritional parameters, such as serum prealbumin, retinol-binding protein, and insulin-like growth factor (IGF) [293].

9.4

Practical recommendations

According to the updated S2k guideline of the German Society for Digestive Metabolic Diseases, zinc substitution is recommended in the case of proven zinc deficiency (blood/plasma < 0.66 mg/mL (66 µg/dL) or < 11.0 µmol/L) though no specific information on dosage and duration is given. The guideline only recommends a maintenance dose of 50 mg of elemental zinc daily after zinc levels have normalized [294].

In patients with liver cirrhosis or diabetes mellitus, measurement of serum/plasma zinc concentration is recommended even before symptoms arise, as zinc concentrations are decreased in these conditions [1,264]. If reduced zinc concentrations are demonstrated reproducibly, zinc supplementation is medically indicated. The treatment with zinc should occur in a controlled way, with regular tests every 6–8 weeks (Table 3). The reference range for serum zinc is 11 - 23 µmol/L or 60 - 120 µg/dL.

Table 3.

Recommended dose of zinc supplements for different degrees of zinc deficiency confirmed by decreased serum zinc concentration (ZC).

ZC (µmol L^-1)	Recommended Dose (Elemental Zn, daily over 6 weeks)
11–9.5	10–15 mg
below 9.0	30 mg
below 6.0	up to 45 mg

Based on many years of experience of one of us (KG), the dosage of a zinc preparation should depend on the extent of the reduction in zinc levels [1,264]. With marginally low levels of zinc (about < 10 µmol/L, 60–80 µg/dL per American College of Gastroenterology, ACG), a daily zinc dose of 15 mg, pending laboratory monitoring after six weeks, is sufficient. Serum zinc levels below 9 µmol/L (below 60 µg/dL per ACG) require zinc doses up to 30 mg of zinc per day. Patients with significantly decreased levels below 6 µmol/L should receive 45 mg zinc per day [1,264].

Zn-histidine or Zn-aspartate are the best absorbed zinc compounds in patients suffering from chronic liver diseases [295]. It is recommended that the zinc supplement is taken 1 h before or after meals to prevent reduced zinc absorption in the gut caused by complexation of zinc by phytate or other components of plant-based food [295]. After a normalization of the blood zinc level, zinc supplementation can be paused. Yet, blood/plasma zinc concentrations should be checked regularly at 8-week intervals, because replenishing zinc stores in bones, liver and muscles can take up to six months. Further details can be found in the literature [295].

Zinc sulfate has good bioavailability but leads to nausea and vomiting in overdoses. Zinc histidine also has increased bioavailability of zinc. The tolerability is very good. Zinc aspartate has similarly good qualities as zinc histidine [296,297]. Polaprezinc (Zinc L-carnosine) has good antioxidant and antiulcerative properties with a significant dose-response relationship with serum zinc in patients with zinc deficiency and improvements in albumin and other parameters in chronic liver disease [298,299].

The ligand in the zinc complex, minimally, has two important effects: it influences bioavailability, and, in some cases, it can have independent supportive pharmacological properties or confer additional biochemical properties to the complex. The latter effect has been demonstrated in the treatment of alcohol-induced liver injury in mice [300].

9.5

Side effects

The main side effects associated with high doses of zinc are interactions with the absorption of calcium, magnesium, iron, manganese, copper and selenium. The most serious interactions are with copper and iron [295]. Iron supplementation should be avoided during zinc therapy [295]. Dietary iron has little interference with zinc. Secondary copper deficiency develops with high oral doses of zinc, e.g., > 50 mg/d zinc for > 10 months [295]. However, shorter periods (6 weeks) using 50 mg/d “elemental” zinc do not seem to cause hypocupremia in healthy individuals. Symptoms of zinc “toxicity” are non-specific and include abdominal pain, diarrhea, dizziness, nausea, vomiting, and in some cases recurrent fever [301].

10

Conclusions

Hippocrates` aphorism "All disease begins in the gut" is gaining specific meaning with recent insights regarding the importance of our gut microbiome in the balance between health and disease. In this article, we focused on the gut-liver axis, which is the central segment in the link from the microbiome to all other gut-organ axes. Dysbiosis of the microbiota and a leaky and inflamed gut are syndromes to be considered in the diagnosis and treatment of liver disease. The essential micronutrient zinc features prominently in these processes.

The effects of zinc deficiency are pleiotropic and affect many metabolic and signaling pathways. Importantly, it is not only dietary zinc itself that needs to be considered in the context of disease, but also a perturbation of the many proteins that control cellular zinc homeostasis and cause zinc dyshomeostasis.

Zinc deficiency is a serious condition. It affects the proper functioning of the gut and liver, and through this axis other organs, and causes a dysbiosis of the gut microbiota [302,303], which can lead to a "leaky gut" and exposure of the liver to harmful substances that can cause systemic inflammation. Therefore, it is a pathogenetic factor in all stages of liver disease. Both the gut and the liver have central roles in controlling systemic zinc homeostasis, and their interaction is the basis for one diseased organ affecting the other. Zinc deficiency needs to be diagnosed, the zinc status monitored, and zinc supplementation implemented as a therapeutic modality if the patient is zinc deficient. Recommendation of dose depends on the degree of zinc deficiency, evaluation of the diet of patient, e.g., the presence of antinutrients such as phytate, and the normalization of serum/plasma levels, which can take time due to the slow exchange rate of some depleted stores.

This year, the American College of Gastroenterology (ACG) published recommendations for nutrition in patients with liver disease [304]. Among the 17 key concepts for nutrition and an extensive discussion of scientific evidence, the document states that "zinc supplementation should be considered for patients with liver disease and hypozincemia and/or signs/symptoms of zinc deficiency". However, the recommendations are vague regarding dosage, duration, zinc compound given, and follow-up with clinical markers. "If used, zinc supplementation at a dose < 50 mg elemental zinc orally per day should be taken with a meal to decrease the potential side effects of nausea and copper deficiency". A "management algorithm" for zinc supplementation has been given previously [305].

Despite the strong evidence for the role of zinc and zinc deficiency in gut and liver disease, the scientific evidence for efficacy of zinc supplementation as a pharmacological intervention is incomplete. Additional well-designed studies with clear comparable directives regarding study design are required. Studies of the impact of zinc supplementation on liver cirrhosis are extremely heterogeneous in design, patient selection, complications, medication, formulation of the zinc supplement, dosage, duration of treatment, timing of lab tests, etc. Moreover, the recommendations clearly show that monotherapy with zinc is not the answer. Other dietary factors are necessary for zinc to function. For example, a condition such as oxidative distress will prevent restoring cellular zinc levels and proper use. The situation will likely not change soon because it is unethical to carry out comparative experiments with patients not receiving zinc. Therefore, with the philosophy of medical care heavily focused on evidence-based medicine, we need to rely on the overwhelming biochemical evidence for the cellular functions of zinc as presented in this article and the experience of the clinician [306]. Thus, one of us (KG) has 30+ years of experience in treating patients with several liver diseases with zinc histidine (Zinkamin, Falk) or zinc aspartate (Unizink 50, Koehler Pharma GmbH) according to the regime presented here. A pinch of zinc indeed does matter significantly in liver disease [29].

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors contributions

KG, DR, WM were involved in conceptualization and writing.

Uncited reference

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