Table 3.

Zinc transport studies using in vitro intestinal models.

Cell Model	Incubation Parameter	Type of Zinc	Main Outcome	Reference
Caco-2 cells Cultivation time: 14 d 3D Transwell (PC membrane) 14 d	ZnCl2 20 µM (Kinetic 0–50 min) 0–100 µM (10 min) (in salt buffer on apical and basolateral side) Inhibitor: ouabain, vanadate, dinitrophenol, sodium cyanide, ammonium vanadate Potential zinc ligands: histidine, cysteine, proline, glutathione	radioactive zinc (65Zn)	-cellular zinc uptake is saturable process -Km = 41 µM Vmax = 0.3 nmol/cm2/10 min -basolateral zinc uptake was partially inhibited (30%) by ouabain and vanadate, which suggests an involvement of the (Na-K)-ATPase in serosal uptake -apical zinc uptake was not affected by metabolic inhibitors and ligands -basolateral zinc uptake (50 min) ~ 0.47 nmol/cm2 -zinc transport ~ 0.8 nmol/cm2 (20 µM, after 50 min) -transport from basolateral to apical is higher than from the apical to the basolateral compartment	[66]
Caco-2 cells Cultivation time: 18–21 d 3D Transwell	ZnSO4 10–1000 µM (for 90 min) 10 nM 1α,25-dihydroxyvitamin D3 (preincubation for 72 h) + 100 µM ZnSO4 (for 90 min) Apical: MES-buffer with NaCl, KCl, MgSO4, CaCl2, glutamine, glucose, Basolateral: 2.5 mg/mL BSA in Hepes with NaCl, KCl, MgSO4, CaCl2, glutamine, glucose,	radioactive zinc (65Zn)	-saturable zinc uptake kinetic up to 1000 µM -Km = 226 µM -zinc transport rate (after 90 min): ~10 µM: ~0.12 nmol/cm2 ~50 µM: ~0.25 nmol/cm2 -zinc transport increased in vitamin D3 incubated cells -mt-2a mRNA and protein was increased with greater zinc concentrations -Crip mRNA (30% less expressed in Caco-2 cells than in rat mucosa) was decreased by vitamin D3 treatment	[238]
Caco-2 cells Cultivation time: 21 d 2D, 3D Transwell (PE membrane)	zinc species: n.a. 1–200 µM (in DMEM + 10% FCS on apical and basolateral side) for 0–30 h	radioactive zinc (65Zn)	-saturable zinc uptake at the basolateral membrane -apical zinc uptake and zinc transport, both from apical to basolateral and vice versa, were not saturable -higher transport from apical to basolateral -transport rate 50 µM: 6 pmol/h/cm2 -transport from apical to basolateral was independent from basolateral zinc concentration -study indicates that zinc uptake and transcellular movement are different at the apical and basolateral side	[67]
Caco-2 cells Cultivation time: 14–16 days of 3D Transwell (Polyethylene terephthalate membrane)	ZnSO4 0–1000 µM (in DMEM + 10% FCS on apical) and 0–450 µM (in DMEM + 10% FCS on basolateral side) for 24 h	total Zn	-applied 0–1000 µM zinc on apical or 7–450 µM zinc basolateral side -transport occurs from both sides to the other compartment -accumulation in the cells was low, particularly when zinc was added on the apical side -zinc toxicity on cell viability and integrity of the intestinal barrier (TEER) 0–2000 µM zinc: -observed higher toxicity when adding high zinc concentrations to the basolateral side	[68]
Caco-2 cells Cultivation time: 18–21 days of 3D Transwell (PC)	ZnCl2 50–200 µM (in serum free medium on apical and basolateral side) for 6 h, 12 h, and 24 h	radioactive zinc (65Zn)	-zinc transport an MT secretion (HPLC analysis) -this study suggest that MT is secreted into the gastrointestinal lumen and plays a role in intestinal zinc uptake -zinc transport (after 6 h) -100 µM: ~2.0 nmol/cm2	[120]
Caco-2 cells Cultivation time: 21 d 3D (PES-HD membranes)	ZnSO4 5 µM or 25 µM (in DMEM + 10% FCS on apical and basolateral) (preincubation for 7 d)	radioactive zinc (65Zn)	-zinc uptake and transport were measured in both apical (AP) and basolateral (BL) directions -rate of apical zinc uptake and transport rate to basolateral was lower in cells pretreated 25 µM zinc -basolateral zinc release was higher in cells treated with 25 µM -cellular zinc uptake 2–3 nmol mg−1 protein -induction of MT (analyzed using radiolabeled cadmium) was zinc-dependent, increasing with zinc concentration	[239]
Caco-2 cells Cultivation time: 21 d 3D Transwell (PC)	ZnSO4 15.6–500 µM (apical: KHB buffer, basolateral: KHB-buffer + 5% BSA)	total Zn	-comparison with zinc transport across isolated rat small intestine -rat: Km = 10–12.1 µM -Caco-2 Km = 11.7 µM Vmax = 31.8 pmol min−1 cm−2 -transport across Caco-2 monolayers is carrier-mediated and energy-dependent -zinc transport into basolateral chamber followed a saturated process -transport rate: 50 µM: 39 pmol min−1 cm−2 -mRNA expression of zip-4, zip-5, znt-1, mt1, mt2 in duodenum, jejunum, and ileum of isolated rat small intestine	[71]
Caco-2 cells Cultivation time: 17 days 3D Transwell (Polytetrafluoroethylene)	ZnSO4 100 µM (serum free medium on apical and basolateral side) for 3–24 h 1 µM hepcidin	stable zinc isotope (67Zn)	-hepcidin reduces basolateral zinc export by post-translationally downregulation of ZnT-1 -cells incubated with hepcidin showed less zinc export while cellular zinc and mt-1a mRNA increased, cell surface ZnT-1 as well as ZnT-1 protein decreased -hepcidin might play a role in controlling zinc absorption and enterocyte subcellular zinc pools	[156]
Caco-2/HT-29-MTX co-culture Cultivation time: 21 days 3D Transwell (PC)	ZnSO4 0–100 µM (apical: serum-free transport buffer, basolateral: DMEM +10% FCS + 0 or 30 mg mL−1 BSA) for 8 h	total Zn	-albumin has a role in in vitro zinc absorption as a basolateral zinc acceptor -cellular uptake is not significantly different with or w/o basolateral added albumin -basolateral serum albumin enhances cellular zinc export to the basolateral side -fractional absorption (25–100 µM): w/o BSA: ~2% with BSA: 5.8–2.9% -zinc transport rates (0–100 µM): w/o BSA: 0.1–2.2 nmol cm−2 with BSA: 1.1–3.6 nmol cm−2	[102]
Caco-2/HT-29-MTX co-culture and Caco-2 monoculture Cultivation time: 21 days 3D Transwell (PC)	ZnSO4 0–100 µM (apical: serum-free transport buffer, basolateral: DMEM + 10% FCS + 30 mg mL−1 BSA) for 4 h	total Zn	-intestinal mucins influence cellular zinc uptake and zinc transport -results suggest that mucins facilitate zinc uptake into enterocytes and act as a zinc delivery system -mucins are an integral part of intestinal zinc absorption -fractional absorption (25–100 µM): monoculture: 1.6–0.9% co-culture: 4.2–1.9% -zinc transport rates (0–100 µM): monoculture: 0.3–1.3 nmol cm−2 co-culture: 1.1–2.3 nmol cm−2	[103]

3D, three-dimensional. BSA, bovine serum albumin. DMEM, Dulbecco’s Modified Eagles Medium. FCS, fetal calf serum. HBSS, Hank’s Balanced Salt Solution. HD, high density. KHB, Krebs-Henseleit buffer. n.a., not available. PC, polycarbonate. PE, polyethylene. PES, polyester. Zn, zinc.