Zinc levels in ASD

The Zn²⁺ status in an individual can be assessed using body fluids, such as urine, plasma, serum, red blood cells, whole blood, or hair^{(Reference Saghazadeh, Ahangari, Hendi, Saleh and Rezaei89)}. The levels of Zn²⁺ assessed in these samples are highly variable among ASD-C^{(Reference Alsufiani, Alkhanbashi, Laswad, Bakhadher, Alghamdi and Tayeb12,Reference Babaknejad, Sayehmiri, Sayehmiri, Mohamadkhani and Bahrami13,Reference Faber, Zinn, Kern and Skip Kingston90–Reference Crăciun, Bjørklund, Tinkov, Urbina, Skalny and Rad93)} . In a systematic review comparing Zn²⁺ levels between individuals with ASD and typically developing (TD) individuals, 36% of the studies reported significantly lower levels of Zn²⁺, with the remaining studies indicating a lower presence of Zn^{2+(Reference do Nascimento, Oliveira Silva, de Morais and de Rezende94)}. Two case-control studies from China found significantly lower blood Zn²⁺ levels among ASD-C than among TD children^{(Reference Guo, Li, Zhang, Chen, Dai and Liu95)}. In addition, associations between decreased serum Zn²⁺ concentrations (≤ 1.28 × 10⁻⁵ mol/L) and an increased risk and severity of ASD, indicating a potential role of Zn²⁺ in the pathophysiology of ASD^{(Reference Wu, Wang, Yan, Jia, Zhang and Han96)} were identified. A meta-analysis of twenty-six studies further confirmed significantly lower blood levels of Zn²⁺ in individuals with ASD (n = 513) than in controls (n = 333), with a mean difference of −0·361 (95% CI: −0·668 to −0·055) in 50% of the investigations^{(Reference Saghazadeh, Ahangari, Hendi, Saleh and Rezaei89)}. Lower serum Zn²⁺ levels and copper toxicity [low Zn²⁺ to Cu²⁺ ratio of 0·618 (95% CI: 0·32–0·99)] were reported in ASD-C in the USA^{(Reference Faber, Zinn, Kern and Skip Kingston90)} and in the Jilin Province, China, with a ratio of 0·64 (95% CI: 0·52–0·75) in ASD-C v. 0·74 (95% CI: 0·59–0·88) in TD children, (p = 0·000)^{(Reference Feng, Shan, Miao, Xue, Yue and Jia97)}. Those with lower [Zn²⁺/Cu²⁺] ratios fared higher on the Autism Behavior Checklist severity scale^{(Reference Feng, Shan, Miao, Xue, Yue and Jia97)}. Similarly, Zn²⁺ levels and the Zn²⁺/Cu²⁺ ratio were significantly lower ([1.36 × 10⁻⁵ mol/L v. 1.44 × 10⁻⁵ mol/L, p = 0·032] and [0·66 ± 0·02 v. 0·76 ± 0·02, p = 0·002], respectively) in 2–9-year-old Bangladeshi ASD-C^{(Reference Siddiqi, Begum, Shahjadi, Afroz, Mahruba and Parvin98)}; however, Cu²⁺ levels (p = 0·020) and ceruloplasmin levels in ASD-C were significantly higher (2.36 × 10⁻⁶ mol/L v. 2.20 × 10⁻⁶ mol/L, p = 0·045) than that of TD children. On the basis of the Chi-squared results, ASD was significantly associated with Zn²⁺ deficiency and Cu²⁺ toxicity (χ ² = 59·239, p = 0·000 and χ ² = 4·313, p = 0·05, respectively)^{(Reference Siddiqi, Begum, Shahjadi, Afroz, Mahruba and Parvin98)}. However, serum Zn²⁺ levels obtained from a large cohort of Irish children were mostly in the normal range, with no significant mean difference [0·06 μmol/L (95% CI: −0·56 to 0·67)] between participants with ASD (11·68 ± 1·7 μmol/L) and controls (11·63 ± 2·1 μmol/L)^{(Reference Sweetman, O’Donnell, Lalor, Grant and Greaney32)}. Similarly, nonsignificant differences in plasma Zn²⁺ levels (1.61 × 10⁻² mol/L) were reported in Brazilian ASD-C^{(Reference Saldanha Tschinkel, Bjørklund, Conón, Chirumbolo and Nascimento99)} as typical reference values are 1.10 × 10⁻² – 1.76 × 10⁻² mol/L^{(Reference Alves, de Brito, Vermeulen, Dantas Lopes, França and Bruno91)}. These discrepancies may be either geographically dependent, with different levels of Zn²⁺ in the soil^{(Reference Hawari, Eskandar and Alzeer100)}, or due to limitations in using plasma/serum concentrations as reliable indicators of Zn²⁺ deficiency. The sensitivity and specificity of plasma samples are limited owing to fluctuations in concentrations resulting from fasting, brief abnormal food intake, circadian variations, and inflammation^{(101,Reference Wieringa, Dijkhuizen, Fiorentino, Laillou and Berger102)} . Moreover, most Zn²⁺ circulates in the blood bound to albumin. Thus, low blood levels of albumin also reduce the plasma and serum concentrations of Zn^{2+(Reference Wieringa, Dijkhuizen, Fiorentino, Laillou and Berger102,Reference Roohani, Hurrell, Kelishadi and Schulin103)} . Alternatively, the assessment of Zn²⁺ levels using hair samples offers a more reliable indication of long-term Zn²⁺ imbalance compared with whole blood or plasma^{(Reference Lowe, Fekete and Decsi104,Reference DiBaise and Tarleton105)} . A cross-sectional study in Italy found low levels of Zn²⁺ in the hair, correlating negatively with ASD severity as categorized by the behavioral scales of play and creativity (r = −0·39; p = 0·006)^{(Reference Fiore, Barone, Copat, Grasso, Cristaldi and Rizzo106)}. Similarly, another study found a significant negative correlation between Zn²⁺ levels and fear and nervousness (r = −0·345; p = 0·022) and between Zn²⁺ levels and verbal communication (r = −0·359; p = 0·017)^{(Reference Blaurock-Busch, Amin, Dessoki and Rabah107)}. Zn²⁺ levels in the hair were significantly lower in Asian individuals with ASD (n = 236) than those of controls (n = 306), with a mean difference of −1·493 (95% CI: −2·43 to −0·56; p = 0·002). Zn²⁺ concentrations in the hair of 29·7% ASD-C in Japan were below −2 standard deviations of the control reference range (86·3–193 parts per million [ppm])^{(Reference Yasuda, Yoshida, Yasuda and Tsutsui108)}. However, among non-Asian participants, the Zn²⁺ levels in hair were significantly higher in individuals with ASD (n = 257) than in the controls (n = 258), with a mean difference of 10·384 (95% CI: 0·04–20·72; p = 0·049)^{(Reference Saghazadeh, Ahangari, Hendi, Saleh and Rezaei89)}. Thus, inconsistent results have been reported. A recent review of studies investigating Zn²⁺ level disparities between individuals with ASD and TD controls concluded that significant differences in the Zn²⁺ concentrations in blood samples (serum and plasma) were identified, and no such disparities were found in hair or urine samples^{(Reference Kaczmarek, Dobrzyńska and Drzymała-Czyż109)}. As mentioned above, Zn²⁺ status is often linked to Cu²⁺ status, where a deficiency in one can lead to toxicity in the other, both affecting human neurodevelopmental and physiological functioning^{(Reference Bjørklund110,Reference Baj, Flieger, Flieger, Forma, Sitarz and Skórzyńska-Dziduszko111)} . Overall, a low Zn²⁺/Cu²⁺ ratio is prevalent in ASD^{(Reference Bölte, Girdler and Marschik78,Reference Faber, Zinn, Kern and Skip Kingston90,Reference Li, Wang, Bjørklund, Zhao and Yin92,Reference Crăciun, Bjørklund, Tinkov, Urbina, Skalny and Rad93,Reference Bjørklund110–Reference Ramaekers, Sequeira, Thöny and Quadros112)} and has been proposed as a candidate diagnostic biomarker for ASD^{(Reference Faber, Zinn, Kern and Skip Kingston90,Reference Li, Wang, Bjørklund, Zhao and Yin92)} . Collectively, a number of studies suggested that Zn²⁺ levels along with Zn²⁺/Cu²⁺ ratios are altered in ASD, although some inconsistencies have also been reported. Therefore, a comprehensive understanding of the role of different factors causing Zn²⁺ deficiency could be valuable in establishing a correlation between Zn²⁺ dyshomeostasis and ASD.

Role of zinc in gut physiology

Approximately 1·5–3 g of Zn²⁺ is present in the human body, mostly in its dissociated form^{(Reference Gropper, Smith and Carr113)}. Zn²⁺ is mainly stored in the skeletal muscles, bone, liver, and skin^{(Reference To, Do, Cho and Jung114,Reference Ross, Hernandez-Espinosa and Aizenman115)} . However, it is also found in the brain, where its abundance is second only to iron in terms of essential trace minerals^{(Reference Ross, Hernandez-Espinosa and Aizenman115–Reference DeBenedictis, Raab, Ducie, Howley, Feldmann and Grabrucker117)}. Therefore, it is not surprising that Zn²⁺ dyshomeostasis in the brain could result in neurodevelopmental and mood disorders^{(Reference Ross, Hernandez-Espinosa and Aizenman115,Reference Gower-Winter and Levenson118)} . Zn²⁺ cannot be stored in the human body beyond the body’s needs. Therefore, daily dietary Zn²⁺ intake is essential to maintain appropriate levels in the body. Rich dietary sources of Zn²⁺ include oysters, red meat, and poultry from animal sources, and whole grains, beans, and nuts from plant sources⁽¹¹⁹⁾. However, the presence of dietary phytate hinders Zn²⁺ absorption because the phosphate group forms strongly insoluble complexes with Zn²⁺, rendering it biologically unavailable^{(Reference Lönnerdal120)}. Indeed, the average Zn²⁺ absorption was reduced from 50% in highly refined diets to approximately 15% in unrefined diets^{(Reference Roohani, Hurrell, Kelishadi and Schulin103)}. On average, Zn²⁺ absorption is approximately 33%^{(Reference Roohani, Hurrell, Kelishadi and Schulin103)}; however, it can increase to 90% in cases of limited Zn²⁺ availability^{(Reference Hara, Takeda, Takagishi, Fukue, Kambe and Fukada15)}. Fig. 2 illustrates Zn²⁺ processing in the human gastrointestinal (GI) system and highlights the factors that influence Zn²⁺ absorption^{(Reference Maret and Sandstead14,Reference Lönnerdal120)} .

Figure 2. Zn²⁺ metabolism in the human body. (a) Release of Zn²⁺ from nucleic and amino acid-bound complexes from food sources. (b) Zn²⁺ absorption through an enterocyte from the apical side through (1) ZIP4, (2) other routes, or (3) paracellular absorption. Zn²⁺ release from the enterocyte to the circulation occurs through ZnT1 transporter. In the blood, Zn²⁺ is carried by albumin (∼70%), macroglobulin (∼30%), or transferrin (∼10%). In the intracellular compartment, Zn²⁺ may be stored as part of metallothionein, which increases in Zn²⁺ supplementation and decreases with deficiency. (c) Zn²⁺ excretion through (1) feces, or (2) other routes.

Zn²⁺ absorption and secretion are controlled by two different transporter families: the zinc importer family (ZIP) and the zinc transporter family (ZnT)^{(Reference Marger, Schubert and Bertrand116)}. The mammalian genome encodes fourteen ZIP and nine ZnT transporters^{(Reference Hara, Takeda, Takagishi, Fukue, Kambe and Fukada15)}. The ZIPs are influx transporters that transport Zn²⁺ from extracellular fluids or intracellular compartments into the cytosol, whereas the ZnTs mostly act as efflux transporters and reduce cytosolic Zn²⁺ levels^{(Reference Hara, Takeda, Takagishi, Fukue, Kambe and Fukada15)}. Zn²⁺ is absorbed throughout the small intestine, primarily in the duodenum and jejunum^{(Reference Maares and Haase121)}. ZIP4, which is located in the apical membrane of the enterocytes, is the main transporter involved in the uptake of Zn²⁺ from the lumen of the gut^{(Reference Fukada, Yamasaki, Nishida, Murakami and Hirano17)}. The primary mode of intestinal Zn²⁺ absorption is carrier-mediated transport mechanisms^{(Reference Roohani, Hurrell, Kelishadi and Schulin103)}, which are mediated by the ZRT and IRT-like protein 4 (ZIP4), encoded by the SLC39A4 gene^{(Reference Jeong and Eide122)}. ZIP4 is regulated by dietary Zn²⁺ intake, where it undergoes rapid degradation with high Zn²⁺ intake to downregulate its absorption^{(Reference Hashimoto and Kambe123)}. ZIP4-mediated regulation is essential for maintaining Zn²⁺ homeostasis with minimal contributions from other carrier proteins, including divalent metal transporter-1, amino acid transporters, and ZIP11, particularly under conditions of Zn²⁺ restriction and paracellular diffusion^{(Reference Hashimoto and Kambe123,Reference Martin, Aydemir, Guthrie, Samuelson, Chang and Cousins124)} . In particular, ZIP11 expression is significantly increased in the colonic tissues to enhance absorption efficiency^{(Reference Martin, Aydemir, Guthrie, Samuelson, Chang and Cousins124)}. In enterocytes, metallothionein acts as both a transient Zn²⁺ storage protein and a carrier to Zn²⁺-requiring enzymes^{(Reference Meguid, Bjørklund, Gebril, Doşa, Anwar and Elsaeid125)}. Both metallothionein and Zn²⁺ transporter 1 (ZnT1) control the release of Zn²⁺ in the portal circulation from duodenal and jejunal cells^{(Reference Cousins126)}. ZnT1 is located in the basolateral membrane of enterocytes and acinar cells, and represents the major transporter controlling the efflux of ions toward the portal circulation^{(Reference Cousins126)}. Urinary and surface loss of Zn²⁺ via sweat represents other routes of Zn²⁺ excretion^{(Reference Roohani, Hurrell, Kelishadi and Schulin103)}, both of which are adjusted during periods of Zn²⁺ depletion or abundance^{(Reference Milne, Canfield, Mahalko and Sandstead127,Reference Solomons and Caballero128)} . The expression of Zn²⁺ transporters is influenced by various factors, and their malfunction causes Zn²⁺ dyshomeostasis and progression of related conditions^{(Reference Kambe, Tsuji, Hashimoto and Itsumura16)}. For example, chronic ethanol exposure alters hepatic Zn²⁺ transporters via oxidative stress, ultimately leading to decreased Zn²⁺ levels in the liver^{(Reference Sun, Li, Zhong, Zhang, Sun and Tan129)}. In mouse and human pancreatic islet cells, hypoxia decreases the expression of the Zn²⁺ transporter 8 and cytosolic Zn²⁺ levels^{(Reference Gerber, Bellomo, Hodson, Meur, Solomou and Mitchell130)}. Several hormones and cytokines play roles in maintaining systemic and cellular Zn²⁺ homeostasis. Melatonin regulates the levels of the Zn²⁺ transporters in the duodenal, jejunal, and ileal tissues (ZnT2, ZIP2, and ZIP4, respectively) to enhance Zn²⁺ absorption^{(Reference Baltaci and Mogulkoc131,Reference Unal, Baltaci, Mogulkoc and Avunduk132)} . Hepcidin is a regulator of iron metabolism; however, it significantly reduces the availability of cellular ZnT1, thereby inhibiting Zn²⁺ export^{(Reference Hennigar and McClung133)}. Estrogen modulates the expression of ZnT3 and Zn²⁺ in hippocampal synapses^{(Reference Lee, Kim, Hong, Lee, Cherny and Bush134)}. Exposure to pro-inflammatory cytokines (particularly interleukin (IL)-1β) down-regulates Zn²⁺ transporters in pancreatic islet cells^{(Reference Egefjord, Jensen, Bang-Berthelsen, Petersen, Smidt and Schmitz135,Reference Muayed, Billings, Raja, Zhang, Park and Newman136)} . Other conditions, such as infection and trauma, may disrupt the Zn²⁺ equilibrium, whereby Zn²⁺ is shifted into cellular compartments due to increased demand for protein synthesis and neutralize the free radicals^{(Reference Gammoh and Rink137)}. Zn²⁺ levels are also affected by conditions that alter serum albumin levels, including inflammation-related albumin decline^{(Reference Roohani, Hurrell, Kelishadi and Schulin103,Reference King138)} .

Zn²⁺ deficiency enhances ZIP4 expression in the intestine. Following a 1 day Zn²⁺-deficient diet, ZIP4 showed a more rapid accumulation rate in the jejunum than in the duodenum^{(Reference Hashimoto, Nakagawa, Tsujimura, Miyazaki, Kizu and Goto139)}. However, this adaptive response also increases the uptake of toxic metals, such as cadmium and lead, which may contribute to the severity of ASD^{(Reference Yasuda, Yoshida, Yasuda and Tsutsui108)}. Low levels of Zn²⁺ have been associated with elevated lead levels^{(Reference Wani, Ansari, Ahmad, Parveen, Siddique and Shadab140)}, and lead retention in the brain affects the behavioral performance of animals^{(Reference Bushnell and Levin141)}. Similarly, Zn²⁺ showed a significant inverse association with serum Cadmium concentration. Specifically, a 10% increase in serum Zn²⁺ levels was associated with an approximately 2% decrease in blood cadmium (95% CI: −3·17 to −0·81) and an approximately 4% increase in urinary cadmium (95% CI: 2·14–6·04)^{(Reference Vance and Chun142)}. Thus, disturbances in Zn²⁺ levels are associated with imbalances in other minerals, indicating the significance of Zn²⁺ homeostasis in gut and brain physiology.

Effect of zinc on gut microbiota

Zn²⁺ is an essential trace element for gut commensal microbiota and plays a significant role in the normal functioning of multiple organs and systems in the human body, particularly the immune and endocrine systems. Furthermore, the role of the gut microbiota in regulating the gut–brain axis linked to several neuropsychiatric and neurodegenerative disorders, including ASD, has been proven to be important^{(Reference Shoubridge, Choo, Martin, Keating, Wong and Licinio148)}. However, data on the relationship between GI tract diseases, the gut microbiota, and Zn²⁺ transporters in individuals with ASD are limited.

The gut microbiota can bind mineral particles and pathogens and compete with commensals and enterocytes for Zn²⁺ absorption. Zn²⁺ can affect the balance between pathogenic and commensal strains^{(Reference Scarpellini, Balsiger, Maurizi, Rinninella, Gasbarrini and Giostra22)}. Although Zn²⁺ is an essential micronutrient for bacteria, its excess results in significant toxicity to bacteria^{(Reference McDevitt, Ogunniyi, Valkov, Lawrence, Kobe and McEwan149)}. Zn²⁺ overexposure has been shown to significantly alter gut microbiota, promoting a shift towards harmful bacteria, including pathogenic Escherichia coli varieties. This is likely due to its high bioavailability, which supports the growth of pathogenic bacteria^{(Reference Skalny, Aschner, Lei, Gritsenko, Santamaria and Alekseenko150)}. Animal studies have shown that long- and short-term feeding of mice diets supplemented with Zn²⁺ is associated with alterations in gut bacterial composition at the phylum, genus, and species levels^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. Low, normal, high, and excessive amounts of Zn²⁺ (0, 30, 150, and 600 mg/kg Zn²⁺) were fed to 3-week-old C57BL/6 mice and maintained for either four weeks (short-term Zn²⁺ intervention) or eight weeks (long-term Zn²⁺ intervention) to demonstrate its effects on various developmental stages from birth to puberty^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. Analysis of the relative abundances of the dominant genera revealed distinct clustering of cecal microbiota in mice fed diets containing various levels of Zn²⁺. During short-term Zn²⁺ intervention, the microbiota of mice fed with a low-Zn²⁺ diet was characterized by enrichment of Desulfovibrio, Enterorhabdus, Flavonifractor, Parasutterella, Mucispirillum, Candidatus Saccharimonas, Alistipes, Odoribacter, and Anaerotruncus, and a decrease in the relative abundance of Sphingomonas ^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. A similar pattern has been reported in individuals with ASD, where Desulfovibrio, Flavonifractor, and Alistipes were enriched^{(Reference Kittana, Ahmadani, Al Marzooq and Attlee152)}. The relative abundances of unidentified Lachnospiraceae, Lachnoclostridium, and Bifidobacterium in the control-Zn²⁺ group were higher than those in the high- and excess-Zn²⁺ groups^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. All these bacteria have a protective role in the gut but have been found to be depleted in the ASD-C group^{(Reference Ma, Liang, Dai, Wang, Luo and Zhang153)}. The high- Zn²⁺ diet was associated with the enrichment of Akkermansia, Faecalibaculum, Helicobacter, and Ileibacterium compared with other diets^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. Mice fed with a 600 mg/kg Zn²⁺ diet showed a higher abundance of Dubosiella, Caulobacter, and Bradyrhizobium, and a lower proportion of Romboutsia, Bacteroides, Lactobacillus, and Bifidobacterium ^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. These findings contrasted with typical ASD microbiota profiles, where Akkermansia is usually depleted and Lactobacillus is enriched, suggesting the protective role of a high Zn²⁺ diet^{(Reference Xu, Xu, Li and Li154)}. In addition, the observed high levels of Helicobacter ^{(Reference Bougafa155)} and low levels of beneficial Bacteroides and Bifidobacterium were consistent with known ASD gut microbiota imbalances^{(Reference Xu, Xu, Li and Li154)}. Notably, during short-term intervention, the ratio of Bacteroidetes to Firmicutes was negatively correlated with the Zn²⁺ dosage^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. A low Bacteroidetes:Firmicutes ratio is a typical feature of ASD^{(Reference Kittana, Ahmadani, Al Marzooq and Attlee152)}.

Long-term Zn²⁺ intervention, compared with short-term Zn²⁺ intervention, reversed the distribution of some bacterial genera and led to an increase in the relative abundance of Akkermansia, Blautia, Alloprevotella, and Ruminiclostridium, and a decrease in Thermovirga ^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. The relative abundances of Parasutterella, Helicobacter, Odoribacter, and Ileibacterium in the control-Zn²⁺ group were higher, and the proportions of Dubosiella, Faecalibaculum, and Bifidobacterium were lower than those in the other groups^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. In contrast, long-term high- Zn²⁺ intervention led to a decrease in most genera compared with the other dietary treatments, except for increased levels of Bifidobacterium and Anaeroplasma. Excess Zn²⁺ diet-fed mice showed increased levels of Bacteroides and Intestinimonas and decreased levels of Lactobacillus ^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. Moreover, pathways, including carbohydrate, glycan, and nucleotide metabolism, were decreased by a short-term low-Zn²⁺ diet. Long-term Zn²⁺ use, especially at high-Zn²⁺ doses, suppresses the abundance of short-chain fatty acid (SCFA)-acid-producing genera and their metabolites^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}.

Recently, Zn²⁺ exposure was found to alter gut microbiota composition in an in vitro simulation of the human colon, whereby exposure to nanoparticles of zinc oxide (ZnO) led to dose-dependent changes in the composition and key functional pathways controlled by the gut microbiome^{(Reference Zhang, Zhu, Guo, Gu, Li and Chen156)}. The relative abundance of Bacteroidetes was high, whereas that of Firmicutes was low. Bacterial biodiversity, SCFA production, and antibiotic resistance genes were also reduced^{(Reference Zhang, Zhu, Guo, Gu, Li and Chen156)}. However, the diversity of the gut microbiota recovered after ZnO nanoparticle exposure was discontinued^{(Reference Zhang, Zhu, Guo, Gu, Li and Chen156)}.

Role of zinc in maintaining a healthy gut

Zn²⁺ contributes to GI development, and its deficiency is involved in the etiology of a wide range of GI diseases^{(Reference Skrovanek, DiGuilio, Bailey, Huntington, Urbas and Mayilvaganan157)}. Overall, the evidence indicates that Zn²⁺ is essential for maintaining proper intestinal function and is a significant factor in gut microbiota biodiversity^{(Reference Skalny, Aschner, Lei, Gritsenko, Santamaria and Alekseenko150)}. In addition, Zn²⁺ can modulate gut microbiota to promote the integrity of the intestinal barrier, reduce inflammation, aid the regeneration of the epithelium, and control the permeation of pathogens^{(Reference Xia, Lian, Wu, Yan, Quan and Zhu158)}. Fig. 3 shows the interplay between gut microbiota and Zn²⁺ in maintaining a healthy gut. Inhibition of bacterial translocation into systemic circulation is associated with maintenance of adequate expression of intestinal tight junction (TJ) proteins^{(Reference Ulluwishewa, Anderson, McNabb, Moughan, Wells and Roy159)}.

Figure 3. Interplay between gut microbiota and Zn²⁺ to maintain a healthy gut. (a) In a Zn²⁺-abundant state, bacteria of the following phyla seem to thrive: Firmicutes, Actinobacteria, Verrucumicrobia, Proteobacteria, and Bacteroidetes. (b) In addition, the mucous membranes are intact with healthy enterocytes, and TJ. No inflammation or bacterial penetration is found. (c) In a Zn²⁺-deficient state, a decrease in Thermovirga and increase in Akkermansia, Blautia, Alloprevotella, and Ruminiclostridium were found in the cecum of mice, with an enrichment of Helicobacter hepaticus. (d) Enterocyte inflammation, impaired TJ, and proliferation of neutrophils along with pathogenic bacteria are found in a Zn²⁺-deficient state.

Zn²⁺ critically regulates TJ complexes in the gut epithelium^{(Reference Ulluwishewa, Anderson, McNabb, Moughan, Wells and Roy159)}. The epithelial and endothelial TJ selectively seal the space between the adjacent cells, preventing unregulated paracellular exchange across the epithelial and endothelial barriers^{(Reference Ulluwishewa, Anderson, McNabb, Moughan, Wells and Roy159)}. Exposure to pathogenic bacteria, certain foods, or micronutrients can drastically affect the ability of the TJ to regulate the permeation of nutrients, water, and electrolytes^{(Reference Ulluwishewa, Anderson, McNabb, Moughan, Wells and Roy159)}. Several studies have shown that Zn²⁺ supplementation or deprivation affects the epithelial barrier function, which is attributed to Zn²⁺-mediated TJ modifications^{(Reference Zhong, McClain, Cave, Kang and Zhou160)}.

Zn²⁺ deficiency can cause intestinal hyperpermeability, commonly known as leaky gut, allowing toxins and antigens produced by harmful bacteria to cross the intestinal lumen and enter the bloodstream^{(Reference Al-Ayadhi, Zayed, Bhat, Moubayed, Al-Muammar and El-Ansary161)}. Recent studies have reported that probiotic bacteria, such as Bifidobacterium and Lactobacillus, can enhance the production of TJ proteins, thus reversing leaky gut disorders^{(Reference Al-Ayadhi, Zayed, Bhat, Moubayed, Al-Muammar and El-Ansary161)}. Therefore, maintaining a normal gut microbiota is critical for the development of an effective intestinal barrier^{(Reference Al-Ayadhi, Zayed, Bhat, Moubayed, Al-Muammar and El-Ansary161)}.

Zinc status, altered gut microbiota, and ASD

Alterations in GI microbiota have frequently been reported in ASD. Several studies have indicated that GI pro-inflammatory factors, including bacterial lipopolysaccharides (LPS), can induce an inflammatory response that eventually affects brain development^{(Reference Kirsten, Queiroz-Hazarbassanov, Bernardi and Felicio162,Reference Kalyan, Tousif, Sonali, Vichitra, Sunanda and Praveenraj163)} . Leaky guts have often been reported in ASD^{(Reference Al-Ayadhi, Zayed, Bhat, Moubayed, Al-Muammar and El-Ansary161,Reference Navarro, Pearson, Fatheree, Mansour, Hashmi and Rhoads164–Reference Fiorentino, Sapone, Senger, Camhi, Kadzielski and Buie167)} . It is associated with a reduced abundance of probiotic bacteria, overgrowth of pathogenic bacteria, accumulation of toxic metabolites, and release of pro-inflammatory cytokines, leading to the development of autistic features through alteration of the gut–brain axis^{(Reference Al-Ayadhi, Zayed, Bhat, Moubayed, Al-Muammar and El-Ansary161)}, as postulated in Fig. 4.

Figure 4. Crosstalk between microbiota-derived metabolites, their role in altering brain activity, and the potential impact of Zn²⁺ on the entire process (a, b). SCFAs may reach the CNS through the vagus nerve and play a neuroactive role (b). (c) Accumulation of toxic metabolites (lipopolysaccharides) produced by distinct gut bacteria that promote immune cell recruitment (d) and trigger the release of pro-inflammatory cytokines (e), could be key factors in the development of autistic phenotypes by affecting the synapses in the brain (f) via the gut–brain axis. (g) Zn²⁺ promotes neural communication and reduces inflammation by suppressing immune cell recruitment and reducing the production of pro-inflammatory cytokines. By contrast, low Zn²⁺ status can compromise intestinal barrier integrity and activate pro-inflammatory signaling, resulting in changes in microbiota composition that may aggravate inflammation.

In general, there are few studies on the potential association between Zn²⁺ status and gut microbiota in humans. Previous studies have demonstrated that Zn²⁺ deficiency is associated with reduced gut microbiota biodiversity^{(Reference Sauer and Grabrucker168–Reference Chen, Jiang, Wang, Chen, Tang and Wang170)}. However, unique taxa, associated with Zn²⁺ deficiency, could not be identified because of limited data available from human studies^{(Reference Skalny, Aschner, Lei, Gritsenko, Santamaria and Alekseenko150)}. Restricted access to Zn²⁺ may influence the maternal microbiota composition and, subsequently, its composition in human offspring^{(Reference Sauer and Grabrucker168)}. Furthermore, the offspring of mice with Zn²⁺ deficiency during pregnancy exhibited ASD-like behavior^{(Reference Vela, Stark, Socha, Sauer, Hagmeyer and Grabrucker36,Reference Sauer, Hagmeyer and Grabrucker171–Reference Schoen, Asoglu, Bauer, Müller, Abaei and Sauer174)} . Interestingly, low dietary levels or bioavailability of Zn²⁺ are associated with altered microbiota composition and inflammation in pregnant mice, establishing a link between maternal Zn²⁺ deficiency, altered microbiota composition, and inflammation^{(Reference Sauer and Grabrucker168)}. Consequently, inflammatory processes, such as neuroinflammation, which can arise under altered microbiota composition, may contribute to altered brain development in ASD^{(Reference Sauer and Grabrucker168)}. A recent study showed that Zn²⁺ is a key regulator of GI development, microbiota composition, and inflammation, with relevance in ASD, and indicated that maternal Zn²⁺ deficiency in mice affects the neonatal Zn²⁺ status of the offspring^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)}. In a recent meta-analysis comparing biomedical factors, trace elements, and microbiota biomarkers between children and adolescents with ASD and TD controls, significant differences were observed. Specifically, ASD-C displayed significantly lower Zn²⁺ levels (mean difference = −6·707, 95% CI: −12·691 to −0·722) and altered microbiota composition, including decreased relative abundance of Bifidobacterium (MD = −1·321, 95% CI: −2·403 to −0·238) and Parabacteroides (MD = −0·081, 95% CI: −0·148 to −0·013), alongside elevated levels of Bacteroides (MD = 1·386, 95% CI: 0·717–2·055) and Clostridium (MD = 0·281, 95% CI: 0·035–0·526)^{(Reference Lin, Zhang, Sun, Li, Li and Zhu176)}. Proteomic approaches have revealed that Zn²⁺ deficiency affects several biological processes, including altered GI physiology and pro-inflammatory signaling, resulting in chronic systemic inflammation, neuroinflammation, and abnormal gut microbiota composition similar to that reported in ASD cases in human subjects^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)}. The microbiota composition of mice with prenatal Zn²⁺ deficiency was significantly altered, with an increase in the phyla Actinobacteria, Proteobacteria, and Tenericutes, and a decrease in Firmicutes^{(Reference Sauer and Grabrucker168)}. Overall, it appears that a low maternal Zn²⁺ status during fetal development is sufficient to compromise intestinal barrier integrity and activate pro-inflammatory signaling, resulting in changes in microbiota composition that may aggravate inflammation, mimicking the comorbidities frequently observed in ASD^{(Reference Sauer and Grabrucker168,Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)} . Thus, alterations in microbiota composition combined with abnormal GI physiology and/or morphology may trigger inflammatory reactions. However, the mechanistic links among inflammation, GI and brain abnormalities, and maternal Zn²⁺ status in individuals with ASD remain unclear.

Zinc transporters—SHANK3 crosstalk in ASD

There exists a crosstalk between ZIP4 and SHANK. The SHANK proteins are encoded by SHANK1, SHANK2, and SHANK3 and comprise the multidomain scaffold proteins at the postsynaptic density of glutamatergic synapses. These proteins connect the neurotransmitter receptors, ion channels, and other membrane proteins to the actin cytoskeleton and G protein-coupled signaling pathways. SHANK also plays a role in synaptic formation and dendritic spine maturation in the brain^{(Reference Shi, Redman, Ghose, Hwang, Liu and Ren194)}. Mutations in SHANK3 on chromosome 22 are associated with ASD^{(Reference Uchino and Waga195)}. A study involving pluripotent cells derived from individuals with Phelan–McDermid syndrome (PMDS) and ASD features showed reduced SHANK3 expression^{(Reference Pfaender, Sauer, Hagmeyer, Mangus, Linta and Liebau196)}. Interestingly, decreased expression of the ZIP2 and ZIP4 transporters has also been observed, especially at SHANK3 sites, suggesting abnormal control of Zn²⁺ homeostasis in these individuals^{(Reference Pfaender, Sauer, Hagmeyer, Mangus, Linta and Liebau196)}. It is possible that the Zn²⁺ deficiency observed in the individuals with ASD decreases the expression of the SHANK3, ZIP2, and ZIP4 proteins^{(Reference Levaot and Hershfinkel178)}. Restoration of the SHANK3 levels or downstream mediators in adults may be a useful therapeutic approach for alleviating synaptic and behavioral impairments associated with SHANK3 mutations^{(Reference Monteiro and Feng197)}. As Zn²⁺ deficiency exhausts both Zn²⁺ pools and SHANK3 availability, lifelong co-therapy with Zn²⁺ supplementation may be required to promote scaffold formation and improve synaptic plasticity^{(Reference Hagmeyer, Sauer and Grabrucker65)}. Therefore, it could be hypothesized that individuals with PMDS and ASD caused by Shankopathies, with one intact copy of SHANK3, may benefit from Zn²⁺ supplementation, as elevated Zn²⁺ may drive the remaining SHANK3 to the postsynaptic density and additionally recruit SHANK2, a second Zn²⁺-dependent member of the SHANK gene family^{(Reference Hagmeyer, Sauer and Grabrucker65)}. Zn²⁺ may also exert its effects through direct modulation of SHANK proteins ^{(Reference Hagmeyer, Sauer and Grabrucker65,Reference Arons, Lee, Thynne, Kim, Schob and Kindler198,Reference Grabrucker, Jannetti, Eckert, Gaub, Chhabra and Pfaender199)} , as SHANK3 has a domain called the sterile alpha motif (SAM) to which Zn²⁺ binds. Altered Zn²⁺ levels in pools stored in glutamatergic synaptic vesicles may affect neurotransmitter release, postsynaptic responses, and trans-synaptic coupling^{(Reference Arons, Lee, Thynne, Kim, Schob and Kindler198)}.

The relationship between SHANK, gut microbiota, and ASD

In addition to the brain, SHANK3 is expressed in the gut epithelium, liver, heart, kidneys, and skeletal muscles^{(Reference Sauer, Bockmann, Steinestel, Boeckers and Grabrucker200,Reference Kim, Ko, Jin, Zhang, Kang and Ma201)} . The use of ASD mouse models is crucial for understanding how microbes affect neurodevelopmental conditions and for discovering targets for the treatment of ASD^{(Reference Schoen, Asoglu, Bauer, Müller, Abaei and Sauer174,Reference Sauer, Bockmann, Steinestel, Boeckers and Grabrucker200,Reference Lee, Jung, Vyas, Skelton, Abraham and Hsueh202–Reference Kazdoba, Leach and Crawley204)} . In particular, SHANK3 knockout mice exhibit GI dysfunction, including dysbiotic gut microbiota composition^{(Reference Tabouy, Getselter, Ziv, Karpuj, Tabouy and Lukic205)} and abnormal absorption of trace minerals, such as Zn^{2+(Reference Pfaender, Sauer, Hagmeyer, Mangus, Linta and Liebau196)}. Low enterocyte levels of SHANK3 also reduced ZIP4 gene expression, and consequently, protein expression^{(Reference Pfaender, Sauer, Hagmeyer, Mangus, Linta and Liebau196)}. SHANK3 plays an important role in gut permeability by affecting intestinal barrier function^{(Reference Wei, Yang-Yen, Tsao, Weng, Tung and Yu206)}. The SAM in the C-terminal domain of the SHANK3 proteins contains a Zn²⁺-binding site; thus, SHANK3 requires Zn²⁺ to function properly^{(Reference Wong, Montgomery, Taylor and Grabrucker207)}. The main SHANK3 rodent model that has been widely utilized in ASD research is the Shank3B−/− ex13–16 mouse, in which exons 13–16 of Shank3B−/− are deleted^{(Reference Wei, Yang-Yen, Tsao, Weng, Tung and Yu206,Reference Wong, Montgomery, Taylor and Grabrucker207)} . This effect is similar to knocking out the gene, which causes a loss-of-function of the SHANK3 protein, as observed in human subjects with ASD. Shank3B−/− mice display the loss of several proteins in the postsynaptic structure, similar to those seen in individuals with ASD, and autistic-like-traits, including repetitive behaviors and deficits in social interaction^{(Reference Wong, Montgomery, Taylor and Grabrucker207)}.

Notably, differences in the gut microbiota of Shank3B−/− mice compared with wild-type (WT) littermates have been observed; thus, this mouse strain has been used to explore the interplay between the gut microbiota and ASD^{(Reference Sgritta, Dooling, Buffington, Momin, Francis and Britton208,Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)} . In the same model, Zn²⁺ supplementation (150 ppm) was shown to reverse alterations in bacterial diversity, expression of TJ genes, and genes involved in immune regulation and energy metabolism^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}.

Probiotic treatment with Lactobacillus reuteri ameliorated ASD-like behaviors caused by dysbiosis in the gut microbiota of Shank3B−/− mice^{(Reference Tabouy, Getselter, Ziv, Karpuj, Tabouy and Lukic205)}. This was positively correlated with the expression of the GABA receptor subunits in the brains of these mice^{(Reference Tabouy, Getselter, Ziv, Karpuj, Tabouy and Lukic205)}. Studies have shown that Zn²⁺ supplementation was beneficial in the Shank3B−/− mouse model of ASD, wherein 6 weeks of postnatal supplementation of Zn²⁺ in the diet resulted in the reversal of some of the ASD-related behaviors^{(Reference Fourie, Vyas, Lee, Jung, Garner and Montgomery210)}. Moreover, Zn²⁺ supplementation in pregnant Shank3B−/− mice inhibited the development of ASD-related behaviors in their offspring^{(Reference Fourie, Vyas, Lee, Jung, Garner and Montgomery210)}. This clear link between dietary Zn²⁺ and ASD behavior suggests the possible involvement of Zn²⁺-dependent signaling in the gut–microbiota–brain axis in ASD^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. A recent study explored the changes in the gut microbiota in relation to autistic-like behaviors of Shank3B−/− mice, wherein Zn²⁺ was supplemented in the diet (using 30 and 150 ppm Zn²⁺). Four types of GI samples (ileum, cecum, colon, and fecal) were collected from the WT and Shank3B−/− mice on either control or supplemented Zn²⁺ diets^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. Specific microbial taxa were differentially abundant in the four experimental groups. A total of seventy-four taxa, mostly from the families Muribaculaceae (Bacteroidetes) and Lachnospiraceae (Firmicutes), were significantly and differentially represented between the WT and Shank3B−/− mice fed with Zn²⁺ (30 ppm)^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. Muribaculaceae, Erysipelotrichaceae, and Blautia were more abundant in the WT mice than in the Shank3B−/− mice fed with Zn²⁺. Blautia is particularly important because it produces butyrate, a SCFA that is beneficial for host health owing to its anti-inflammatory effects on the mucosa. It also exerts positive neuromodulatory effects on the gut–microbiota–brain axis, owing to its ability to cross the blood–brain barrier^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209,Reference Mirzaei, Bouzari, Hosseini-Fard, Mazaheri, Ahmadyousefi and Abdi211)} . SCFA may also contribute to lowering the intestinal pH, increasing Zn²⁺ solubility, and thus, increasing the absorption of dietary Zn^{2+(Reference Wong, Montgomery, Taylor and Grabrucker207,Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)} . Among the Shank3B−/− mice, the bacterial Enterobacteriaceae and Coriobacteriaceae families were more abundant in those supplemented with Zn²⁺ diet (150 ppm) than in the control Zn²⁺ diet (30 ppm) mice, which had an increased abundance of Muribaculaceae and Ileibacterium ^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. Diets can alter the host production of bile acids, which can affect the gut microbiome, favoring the proliferation of the bacterial species that metabolize bile acids, such as members of the Coriobacteriaceae^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. This, in turn, can affect gut barrier permeability through the interaction of bile acids with the epithelial cells. Similarly, members of Muribaculaceae, Blautia, and Faecalibaculum were more abundant in WT mice fed a 30 ppm Zn²⁺ diet compared with the Shank3B−/− mice fed with a 150 ppm Zn²⁺ diet, which had a higher abundance of Coriobacteriaceae and Escherichia/Shigella ^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. From the above studies, it is evident that altered Zn²⁺ status can cause dysbiosis by altering microbial composition and diversity. In the presence of dysbiotic microbiota, the overall microbial products change, affecting gut function and the ability to process nutrients^{(Reference Chen, Wang, Wang, Yu, Ding and Wang151)}. This evidence suggested a link between genes associated with ASD, dietary Zn²⁺, and gut microbiota in the pathophysiology of ASD.

Potential role of prenatal zinc deficiency in ASD

Zn²⁺, among other trace minerals, plays a critical role in early neurodevelopment^{(Reference Ross, Hernandez-Espinosa and Aizenman115,Reference Gower-Winter and Levenson118)} . Various factors that direct stem cell proliferation during neurodevelopment require Zn^{2+(Reference Frederickson, Suh, Silva, Frederickson and Thompson181)}. Prenatal Zn²⁺ deficiency (PZD) is considered an environmental risk factor for ASD development in the offspring^{(Reference Grabrucker212)} which has direct and indirect implications^{(Reference Vela, Stark, Socha, Sauer, Hagmeyer and Grabrucker36,Reference Sauer, Hagmeyer and Grabrucker171–Reference Schoen, Asoglu, Bauer, Müller, Abaei and Sauer174)} . Zn²⁺ is supplied to the brain via the blood–brain and blood–cerebrospinal fluid barriers to maintain homeostasis^{(Reference Takeda213)}. This ion is stored in the amygdala and Zn²⁺-abundant glutamatergic neurons, which act as the neuromodulators of synaptic transmission^{(Reference Takeda213)}. The synaptic vesicles store Zn²⁺ at high concentrations and regulate its release into the synaptic cleft after the physiological stimulation of the glutamatergic neurons^{(Reference Zhang, Dischler, Glover and Qin214)}. Therefore, dietary Zn²⁺ deprivation, particularly during early brain development, may influence Zn²⁺ homeostasis by affecting zincergic innervation^{(Reference Sauer, Hagmeyer and Grabrucker171,Reference Zhang, Dischler, Glover and Qin214)} . Low maternal Zn²⁺ levels result in reduced brain Zn²⁺ levels in offspring^{(Reference Grabrucker, Boeckers and Grabrucker172,Reference Grabrucker, Jannetti, Eckert, Gaub, Chhabra and Pfaender199,Reference Grabrucker212,Reference Liu, Adamo and Oteiza215)} . Signaling pathways at the glutamatergic synapses, involving SHANK2 and SHANK3 proteins, are influenced by PZD and linked to ASD^{(Reference Grabrucker212)}. PZD animal models, including mice born to mothers fed a 5-week Zn²⁺ deficient diet (< 5 ppm) prior to mating and during gestation^{(Reference Grabrucker212)} also showed some ASD-like behaviors, such as increased anxiety levels, impaired nest building and vocalizations, and altered social interactions^{(Reference Hagmeyer, Haderspeck and Grabrucker31)}. Additional consequences included the loss of a family of proteins that have a major scaffolding function at the synapse, namely the ProSAP1/SHANK2 and ProSAP2/SHANK3 proteins^{(Reference Grabrucker, Jannetti, Eckert, Gaub, Chhabra and Pfaender199)}, and altered hemisphere-specific distribution, with a significant gain in the right hemisphere in pups (p = 0·017)^{(Reference Grabrucker, Haderspeck, Sauer, Kittelberger, Asoglu and Abaei173)}. Similarly, the PZD and homozygous Shank3B−/− mice showed both convergent and divergent brain region abnormalities^{(Reference Schoen, Asoglu, Bauer, Müller, Abaei and Sauer174)}. Prenatal Zn²⁺ is also crucial for gut formation in offspring^{(Reference Vela, Stark, Socha, Sauer, Hagmeyer and Grabrucker36)}. Indeed, Zn²⁺ deficiency causes the loss of brush border integrity and might lead to the dysfunction of disaccharidases, thereby altering gut maturation^{(Reference Wan and Zhang216)}. The offspring of an adult female swine fed a 250 ppm Zn²⁺ diet showed improved intestinal development^{(Reference Vela, Stark, Socha, Sauer, Hagmeyer and Grabrucker36)}. In addition to GI development, Zn²⁺ is an essential regulator of microbial composition and GI inflammation in ASD^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)}. Proteomic analysis of ileal samples from PZD mice revealed alterations in 183/1010 proteins involved in the pathways regulating the innate immune system, focal adhesions, TJ, and gap junctions^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)}. This suspected dysregulation of gut permeability was evidenced by increased serum Zonulin-1, a protein synthesized in the intestinal cells that regulates intestinal permeability in PZD. Gut inflammation was confirmed by a significant increase in serum inflammatory cytokines (IL1α, p = 0·037; IL1β, p = 0·050; IL7, p = 0·050; and IL17α, p = 0·011) in PZD pups^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)} secondary to neuroinflammation^{(Reference Kirsten, Queiroz-Hazarbassanov, Bernardi and Felicio162)}. The microbiota of PZD mice showed a significant increase in Bacteriodetes and Actinobacteria (p < 0·001 and p < 0·001, respectively) and a significant decrease in Firmicutes (p < 0·001) at the phyla level, whereas at the genus level, PZD mice showed significantly reduced diversity (p < 0·05)^{(Reference Sauer, Malijauskaite, Meleady, Boeckers, McGourty and Grabrucker175)}. Learning and memory impairments have also been reported in rats^{(Reference Hagmeyer, Haderspeck and Grabrucker31,Reference Halas, Hunt and Eberhardt217,Reference Tahmasebi Boroujeni, Naghdi, Shahbazi, Farrokhi, Bagherzadeh and Kazemnejad218)} . Concerning immune system regulation, diets containing < 1 mg/kg Zn²⁺/day appeared to cause thymus atrophy, thereby impacting the T-helper cells and having teratogenic effects, as evidenced by the resulting congenital defects in mouse offspring^{(Reference Sauer, Hagmeyer, Grabrucker, Holick and Nieves219,Reference Uriu-Adams and Keen220)} . Such systemic inflammatory events and prenatal stress have been reported to aggravate Zn²⁺ deficiency in mothers, thereby increasing the risk of ASD in the offspring^{(Reference Sauer, Hagmeyer, Grabrucker, Holick and Nieves219,Reference Uriu-Adams and Keen220)} . Although more controlled studies are warranted to prove the association between PZD and ASD, these studies emphasized the importance of sufficient Zn²⁺ availability, especially during the prenatal period, to ensure proper fetal neurodevelopment and possibly prevent nongenetic causes of ASD. Therefore, it is essential to understand whether Zn²⁺ supplementation can alleviate ASD-related symptoms or reduce the risk of ASD.

Effects of zinc supplementation

Grabrucker et al. advocated Zn²⁺ supplementation as a potential treatment for ASD, citing its essential roles in the neurological, GI, and immune systems, and its ability to reverse behavioral abnormalities observed in rodent models of ASD^{(Reference Indika, Frye, Rossignol, Owens, Senarathne and Grabrucker221)}. Overall, Zn²⁺ supplementation may be useful in young individuals with ASD, as summarized in Table 1. In particular, diarrhea in ASD-C has been linked to Zn²⁺ deficiency, as its supplementation significantly reduces this symptom by potentiating immune function and ameliorating neurosensory deficits associated with Zn²⁺ deficiency^{(Reference Vela, Stark, Socha, Sauer, Hagmeyer and Grabrucker36)}. As reviewed earlier, all the pathways altered by Zn²⁺ deficiency may contribute to the etiology and severity of ASD. Observing the actual effects of Zn²⁺ supplementation in a pediatric population with ASD, a supplementation trial of 12 weeks significantly improved ASD severity according to Childhood Autism Rating Scale (CARS) scores (p = 0·0002) in thirty children, which may be related to the regulation of the previously discussed pathways^{(Reference Meguid, Bjørklund, Gebril, Doşa, Anwar and Elsaeid125)}. Interestingly, there was also a significant improvement in gross motor development (TGMD-2). In addition, metallothionein and serum Zn²⁺ and Cu²⁺ levels were significantly ameliorated, thereby improving the Zn²⁺/Cu²⁺ ratio. An increase in metallothionein, following Zn²⁺ supplementation, was expected and has been previously documented^{(Reference Irato, Sturniolo, Giacon, Magro, D’Inca and Mestriner222)}. However, a reduction in the gene expression of metallothionine 1 (MT1), following dietary Zn²⁺ supplementation, was also observed, warranting further investigation on this association in ASD-C^{(Reference Meguid, Bjørklund, Gebril, Doşa, Anwar and Elsaeid125)}.

Table 1 Effect of Zn²⁺ supplementation on ASD-related features or genes expression in individuals with ASD and animal and cell culture models of ASD

↑: Increase; ↓: Decrease; AMPAR, α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor; ASD, autism spectrum disorder; ASD-C, children with autism spectrum disorder; BDNF, brain derived neurotropic factor; BTBR, Black and Tan Brachyury; CARS, childhood autism rating scale; EGFP, enhanced green fluorescent protein; ppm, parts per million; KO, knockout; LPS, lipopolysaccharide; NMDAR, N-methyl-D-aspartate receptor; NS, not significant; PPR, AMPAR-mediated paired-pulse ratio; SNV, single nucleotide variation; WT, wild-type; Zn²⁺, zinc; ZnSO₄, zinc sulfate.

Most studies of Zn²⁺ supplementation have been conducted using animal models. A six-week experimental trial on Shank3B−/− mice displaying ASD-like characteristics fed with a regular and Zn²⁺ supplemented diet found that Zn²⁺ supplementation prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. These effects were attributed to the fact that the proteins encoded by the SHANK genes are regulated by Zn²⁺ ions, and increased Zn²⁺ levels are associated with improved scaffold protein formation and synaptic maturation^{(Reference Hagmeyer, Sauer and Grabrucker65,Reference Vyas, Lee, Jung and Montgomery223)} . Zn²⁺ supplementation also increased the recruitment of Zn²⁺-sensitive SHANK2 to the synapses. Also, this protein possesses a SAM domain that can bind to Zn²⁺; therefore, it is possible that higher dietary Zn²⁺ intake increases SHANK2 recruitment as evidenced in Shank3B ex13–16−/− mice^{(Reference Fourie, Vyas, Lee, Jung, Garner and Montgomery210)}. SHANK2 is known for its role in the development of Zn²⁺-sensitive synapses and the control of postsynaptic density^{(Reference Vyas, Jung, Lee, Garner and Montgomery224)}. Notably, in vitro studies reported that Zn²⁺ supplementation was able to rescue and increase synaptic density in SHANK2 single nucleotide variation (SNV)-associated deficits and, in turn, their ability to promote synapse formation and maintenance^{(Reference Vyas, Jung, Lee, Garner and Montgomery224)}. Additional outcomes following Zn²⁺ supplementation include reduced synaptic transmission through the NMDA-type glutamate receptors and improvement of the slow decay of NMDAR-mediated currents^{(Reference Fourie, Vyas, Lee, Jung, Garner and Montgomery210)}. The Zn²⁺-mediated mitigation of ASD-like symptoms co-occurs with its modulation of NMDA current, suggesting that the NMDAR may serve as targets for Zn²⁺ dietary changes^{(Reference Fourie, Vyas, Lee, Jung, Garner and Montgomery210)}. Similarly, 150 ppm Zn²⁺ supplementation in another 9–11 week trial using the rodent model of ASD, Tbr1+/− (Tbr1 haploinsufficiency), prevented impairments in auditory fear memory and social interaction and significantly restored the synaptic puncta density of GluN1, which is essential for functional NMDAR as well as SHANK3 expression^{(Reference Lee, Jung, Vyas, Skelton, Abraham and Hsueh202)}. In LPS-exposed pregnant Wistar rats, ASD-like behavioral, brain, and immune disturbances were induced by significantly elevated levels of free-mature BDNF (p < 0·05). Supplementation of rats with Zn²⁺ reduced the elevation of LPS-induced BDNF to the same level as that in the control group^{(Reference Kirsten, Queiroz-Hazarbassanov, Bernardi and Felicio162)}. Furthermore, LPS exposure reduced the number of offspring, and treatment with Zn²⁺ prevented this reduction. Moreover, pups prenatally exposed to LPS spent significantly longer periods (p < 0·05) without calling their mothers, and posttreatment with Zn²⁺ prevented this LPS-induced behavioral impairment^{(Reference Kirsten, Queiroz-Hazarbassanov, Bernardi and Felicio162)}. This indicated that BDNF hyperactivity, as observed in individuals with ASD, may contribute to communication deficits that may be improved by Zn²⁺ supplementation. Moreover, microbial diversity seemed to be affected in those with ASD, and similar features were found in the Shank3B−/− mouse models, where significantly higher microbial diversity was observed in WT than mutated mice (p < 0·05)^{(Reference Wong, Jung, Lee, Fourie, Handley and Montgomery209)}. Overall, Muribaculaceae (Bacteroidetes) and Lachnospiraceae (Firmicutes) were significantly differentially represented between WT and Shank3B−/− mice fed Zn²⁺ (30 ppm) (p < 0·05). An experimental trial reported improved ASD-like characteristics in offspring born from Shank3B−/− mice with maternal Zn²⁺ supplementation and improved social interactions in the offspring^{(Reference Lee, Jung, Vyas, Skelton, Abraham and Hsueh202)}. It is important to note that the rescue effect of Zn²⁺ on social behaviors was maintained throughout adulthood in mice (week 16 of age), indicating the long-lasting benefits of Zn²⁺ supplementation during early brain development^{(Reference Vyas, Lee, Jung and Montgomery223)}. Furthermore, there were no significant differences in excessive grooming behavior in adult mice or anxiety behavior in juvenile and adult mice compared with the offspring born to WT mice^{(Reference Vyas, Lee, Jung and Montgomery223)}. In line with the three diagnostic features of ASD, BTBR T + tf/J (BTBR) mice displayed behaviors consistent with them, such as impaired social interaction and communication, as well as increased repetitive behaviors^{(Reference Meyza, Defensor, Jensen, Corley, Pearson and Pobbe225)}. A recent study compared BTBR mice with the C57BL/6 WT strain for several baseline ASD-like characteristics and then investigated the effect of Zn²⁺ supplementation using 60 ppm of Zn²⁺ in water^{(Reference Zhang, Xu, Ma, Wang, Jin and Li226)}. Specifically, Zn²⁺ supplemented water increased the social preference and social novelty indices in the BTBR mice, as determined by a three-chambered socialization experiment (p < 0·001), indicating improved social behavior. Zn²⁺-supplemented water also significantly improved repetitive behavior in the mice, as indicated by the marble burying and self-grooming tests. The BTBR mice had a significantly higher rate of marble burial than WT mice (p < 0·01) before treatment, which was significantly reduced in the BTBR mice (p < 0·05) post-treatment. In addition, the Holm–Sidak test confirmed that self-grooming time was significantly increased in the BTBR mice compared with that in the C57BL/6 mice (p < 0·001); whereas, self-grooming time was significantly reduced in BTBR mice post-Zn²⁺ treatment (p < 0·001). Finally, Zn²⁺-supplemented water reduced anxiety-like behavior, as confirmed by the open field test. Compared with WT mice, the BTBR mice displayed a more anxious behavior, which was significantly reduced post-Zn²⁺ water treatment (p < 0·05). Moreover, this treatment significantly reduced the convulsion susceptibility of the BTBR mice (p < 0·05) compared with that of the WT mice. It also boosted hippocampal cell growth, indicating that Zn²⁺ supplementation significantly restored neuronal proliferation in the hippocampus of the BTBR mice (p < 0·05). Collectively, these investigations suggested that Zn²⁺ can help mitigate autism-related symptoms in the BTBR model of autism^{(Reference Zhang, Xu, Ma, Wang, Jin and Li226)} and these studies highlighted that Zn²⁺ may play a significant role from gestation throughout adulthood.