Strategies for inducing and validating zinc deficiency and zinc repletion

Abstract

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases.

NEW & NOTEWORTHY This methods paper details dietary approaches to alter zinc homeostasis in rodents and qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases.

INTRODUCTION

Zn²⁺ is the second most abundant trace metal in the human body, constituting 2 to 3 g (1). Skeletal muscle and bone are major Zn²⁺ reservoirs, accounting for approximately 50% and 30% of total body Zn²⁺, respectively (2–5). Lower Zn²⁺ fractions are distributed across various tissues, including the kidney, prostate, liver, gastrointestinal tract, skin, lung, brain, heart, and pancreas (3, 5–8). Within these organs, intracellular Zn²⁺ serves as an essential cofactor for the catalytic activity and structural integrity of over 300 proteins, including those involved in synthesis of macromolecules and cell division (9–12).

Since its initial recognition in 1963 (13), Zn²⁺ deficiency has been increasingly implicated as a hidden culprit in multiple health conditions and chronic diseases such as diabetes, kidney diseases, cancers, neurodegenerative diseases, gastrointestinal disorders, respiratory infections, and skin conditions (5, 14, 15). For more than 60 years, epidemiological, clinical, and experimental studies have focused on determining the role of Zn²⁺ deficiency in the development of these conditions and diseases (1, 5, 13, 14, 16). Given the continued interest in the role of zinc in the onset and progression of diseases, more research is needed to identify the impact of this important micronutrient. The knowledge gained from these valuable studies will advocate for effective approaches that integrate Zn²⁺ supplementation into existing therapeutic strategies that address a plethora of conditions and diseases.

To directly study the impact of this important micronutrient on health and disease, standardized methods are required to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. We also provide qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases.

MATERIALS AND EQUIPMENT

Key resources can be found in Table 1. The following solutions are indicated:

• 1) 
  4% Chelex solution:

  • Suspend Chelex 100 chelating resin (2 g) in double distilled H₂0 (50 mL);
  • on a rocker, shake Chelex solution for 2–3 min;
  • centrifuge Chelex solution at 6,000 rpm for 1.5–2 min;
  • collect clear portion of supernatant and aliquot 10 mL into multiple clear conical tubes; and
  • store 4% Chelex solution aliquots at −80°C until use.
• 2) 
  Tris buffer:

  • In an aliquot of 4% Chelex solution (10 mL), suspend a Tris-buffered saline tablet;
  • using a rocker, gently agitate the buffer until the tablet is dissolved;
  • aliquot Tris buffer (5 mL) into black conical tubes; and
  • store Tris buffer aliquots at 4°C until use.
• 3) 
  Zn²⁺ detector solution:

  • Into an aliquot of Tris buffer (5 mL), dispense Zn detector (25 µL) (note: Zn detector is light sensitive),
  • vortex for 10 s, and
  • immediately use Zn detector solution for zinc quantification.
• 4) 
  ZnCl₂ standards (Table 2):

  • with the reagents provided in the Zinc Quantification Kit, prepare a 1 mM ZnCl₂ standard by suspending 100 mM ZnCl₂ standard (10 µL) into assay buffer (990 µL);
  • to prepare a 100 µM ZnCl₂ standard, suspend 1 mM ZnCl₂ (100 µL) into Tris buffer (900 µL);
  • to prepare the designed concentrations of ZnCl₂ standards, begin with the 100 µM ZnCl₂ standard and perform a serial dilution in Tris buffer; and
  • store ZnCl₂ standards at 4°C until use (note: discard ZnCl₂ standards after two uses).

Table 1.

Key resources

Reagent/Resource	Source	Identifier
Chemicals
Chelex 100 chelating resin	Bio-Rad, Hercules, CA	1421253
Tris-buffered saline table	Millipore-Sigma, MO	T5030-100TAB
Protease inhibitor cocktail	Millipore-Sigma, MO	P8340-1ML
Critical commercial assays
Zinc quantification kit	Abcam, Boston, MA	ab176725
Experimental models
C57BL/6J male mice	The Jackson Laboratory	JAX:000664
Other
Standard chow diet	Teklad, Inotiv, IN	Teklad 2018
Zn2+ adequate diet (50 ppm)	Teklad, Inotiv, IN	TD 85420
Zn2+ deficient diet (1 ppm)	Teklad, Inotiv, IN	TD 85419
Deionized distilled H2O	Millipore-Sigma, MO	6442-88
Purified drinking H2O	Aquafina, Sam’s Club	988708
Black, clear bottom 96-well plate	Thermo Fisher Scientific, Rochester, NY	265301
Conical tubes
Clear (15 mL)	Celltreat, Pepperell, MA	230411-R
Black (15 mL)	Celltreat, Pepperell, MA	229431
Clear (50 mL)	Thermo Fisher Scientific, Rochester, NY	14-432-22
Microcentrifuge tubes
Black	Celltreat, Pepperell, MA	229437
Clear	Electron Microscopy Sciences, Hatfield, PA	63027-02

Table 2.

ZnCl₂ standards

ZnCl2 Standards	Tris Buffer, µL	ZnCl2 Concentration, µM
100 µL of 1 mM	+900	100
500 µL of 100 µM	+500	50
500 µL of 50 µM	+500	25
500 µL of 25 µM	+500	12.5
500 µL of 12.5 µM	+500	6.25
500 µL of 6.25 µM	+500	3.125
500 µL of 3.125 µM	+500	1.56
500 µL of 1.56 µM	+500	0.78
500 µL of 0.78 µM	+500	0.39
500 µL of 0.39 µM	+500	0.195
500 µL of 0.195 µM	+500	0.097
—	+1.000	0

Institutional Permissions

All animal experiments performed were in accordance with the Animal Care and Use Committee of Wright State University and were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The appropriate licenses and permission for animal experiments should be obtained before proceeding with this protocol.

Step-by-Step Method Details

I. Acclimation period.

Timing: 2 wk—Acclimation of mice to Zn²⁺-adequate diet

• Critical: Group house mice at 27°C in a 12-h:12-h light/dark-cycled room.

  • a. Until 6 wk of age, maintain C57BL/6 mice on a standard chow diet.
  • b. 
    At 6 wk old, acclimate mice to a Zn²⁺-adequate (ZnA) diet for 2 wk.

    • i. 
      Provide mice with ad libitum access to food and purified drinking H₂O.

    Note: Since the quality of tap H₂O may vary between facilities, use commercially available purified drinking H₂O.

  • c. Each week, weigh individual mice to monitor growth rate.
  • d. At the end of the acclimation period, proceed to Zn²⁺ depletion period.

II. Zn²⁺ depletion period.

Timing: 10 wk

• Critical: Since mice are social animals, group housing provides a less stressful environment than individually housing mice. If mice must be housed individually, ensure that all experimental groups are housed similarly. To ensure that mice consume similar nutritional content, pair-feed mice by providing the ZnA group with the amount consumed by ZnD mice. Avoid using glass H₂O bottles, as glass may be a source of Zn²⁺. To avoid the consumption of fecal Zn²⁺, weekly mouse cage changes are required.

  • a. 
    Group 1 (Zn²⁺ adequate): For the control group, maintain a subset of the ZnA mice on the ZnA diet for up to 10 wk. Continue to provide ZnA mice with commercially available purified drinking H₂O.
  • b. 
    Group 2 (Zn²⁺ deficient): Switch a subset of ZnA mice to an ingredient-matched ZnD diet for 10 wk. To maintain a Zn²⁺-free environment, provide ZnD mice with deionized, distilled drinking H₂O in plastic containers. Critical: Visual signs of chronic Zn²⁺ deficiency include growth retardation (Fig. 2B), hair loss (Fig. 2C), and loose stool. However, the absence of these visual signs should not disqualify mice from studies. Zn²⁺ levels must be quantified to verify changes in Zn²⁺ levels (see “Zn²⁺ Quantification” Step).Note: While Zn²⁺ deficiency was confirmed at week 10 (Fig. 1), this status may occur at an earlier timepoint. As such, plasma and urine Zn²⁺ levels should be quantified to identify timepoint of changes in Zn²⁺ levels (see “Zn²⁺ Quantification” Step).

Figure 2.

Zn²⁺ homeostasis plays a critical role in growth and development. To investigate the impact of dietary Zn²⁺ restriction and Zn²⁺ repletion on mouse health, mice were fed a Zn²⁺-adequate diet (ZnA, gray) or Zn²⁺-deficient diet (ZnD, red) for 10 wk. At week 8, a subset of mice on the ZnD diet was placed back on the ZnA diet (ZnR, gray with red stripes). Effects on weekly weight gain (A), body mass at week 10 (B), and skin health (C) were examined. *P < 0.05 vs. ZnA; #P < 0.05 vs. ZnD.

Figure 1.

Impact of dietary Zn²⁺ restriction and repletion on Zn²⁺ levels. To probe the influence of dietary Zn²⁺ restriction and Zn²⁺ repletion on Zn²⁺ levels, mice were fed a Zn²⁺-adequate diet (ZnA, gray) or Zn²⁺-deficient diet (ZnD, red) for 10 wk. At week 8, a subset of mice on the ZnD diet was placed back on the ZnA diet (ZnR, gray with red stripes). Effects on plasma (A) and urine (B) Zn²⁺ levels were assessed. *P < 0.05 vs. ZnA; #P < 0.05 vs. ZnD.

III. Zn²⁺ repletion period.

Timing: 2 wk

• c. Group 3 (Zn²⁺ repletion): For Zn²⁺ repletion studies, after 8 wk on a ZnD diet (as described for group 2), return mice to the ZnA diet and purified drinking H₂O (as described for group 1). For the remaining 2 wk of the study, maintain this subset of mice (ZnR) on the ZnA protocol.

IV. Sample collection.

• a. 
  Plasma samples

  • 1. 
    From the submandibular vein, collect blood samples from mice. Note: Collection method should be perfected to avoid hemolysis.
  • 2. 
    Centrifuge collected blood samples at 1,500 g at 4°C for 15 min.
  • 3. 
    Collect and aliquot plasma (20 µL) into clear microcentrifuge tubes.
  • 4. 
    Proceed to “Zn²⁺ quantification” step.

    • Optional step: Store plasma samples at −80°C for up to 12 mo.
• b. 
  Urine samples

  • 1. 
    To collect 24-h urine samples, house mice in metabolic cages.
  • 2. 
    Centrifuge collected urine samples at 2,000 g at 4°C for 10 min.
  • 3. 
    Collect and aliquot debris-free urine (100 µL) into black microcentrifuge tubes.
  • 4. 
    Proceed to “Zn²⁺ quantification” step.

    • Optional Step: Store urine samples at −80°C for up to 12 mo.

V. Zinc quantification.

Timing: [30 min]

Note: Perform all steps in a dark area.

• a. Dispense Tris buffer (30 µL) into appropriate wells of a black, clear bottom 96-well plate.
• b. In duplicate, pipet Zn²⁺ standards (10 µL) and unknown samples (10 µL) into the appropriate wells of the 96-well plate.
• c. To protect Zn²⁺ standards and unknown samples from light, cover 96-well plate with aluminum foil.
• d. With a rocker, gently shake 96-well plate at room temperature for 15 min to ensure the mixing of Zn²⁺ Standards and unknown samples with Tris buffer.
• e. With a fluorescence plate reader, measure background fluorescence (Ex/Em = 485/525) of Zn²⁺ standards and unknown samples.
• f. Dispense Zn detector (30 µL) into the appropriate wells of the 96-well plate.
• g. After covering the 96-well plate with aluminum foil, gently shake on a rocker for 10 s.
• h. Incubate covered 96-well plate at room temperature for 15 min and then measure Zn Detector fluorescence (Ex/Em = 485/525).

VI. Data analysis.

• a. To obtain net fluorescence, subtract background fluorescence from Zn Detector fluorescence.
• b. With the net fluorescence, calculate Zn²⁺ concentration of unknown samples by extrapolating values from the Zn²⁺ standard curve.

EXPECTED OUTCOMES

Impact of Dietary Zn²⁺ Restriction and Repletion on Growth and Development

To assess the impact of Zn²⁺ restriction on nutritional status, body mass was measured in mice placed on a ZnA or ZnD diet for 10 wk (Fig. 2). Until week 5, mice on a ZnD diet had similar body masses as mice maintained on the ZnA diet (Fig. 2A). However, starting at week 6, ZnD mice experienced slower weight gain. Furthermore, between 6–8 wk, some ZnD mice experienced alopecia (Fig. 2C). While average food consumption was not significantly different (14.74 ± 0.21 vs. 15.21 g ± 0.54), at week 10, mice on the ZnD diet had significantly less body mass (25.769 g ± 0.601) compared with ZnA mice (28.08 g ± 0.626) (Fig. 2B).

To assess the impact of Zn²⁺ repletion (ZnR) on nutritional status, at week 8, ZnD mice were returned to a ZnA diet. Zn²⁺ repleted mice experienced an accelerated growth rate (Fig. 2A). After 2 wk, body mass (27.42 ± 0.583 g) and food consumption (15.78 ± 0.39 g) of ZnR mice (Fig. 2B) was comparable to mice maintained on the ZnA diet. These results indicate that chronic dietary Zn²⁺ restriction impairs growth, and acute dietary Zn²⁺ repletion improves development.

Impact of Dietary Zn²⁺ Restriction and Repletion on Plasma and Urinary Zn²⁺ Levels

With the Zn²⁺ Quantification protocol, plasma Zn²⁺ levels were quantified and normalized to mouse body mass (Fig. 1A). At week 10, mice maintained on the ZnA diet had plasma Zn²⁺ levels of 3.19 µM/g body mass. In contrast, plasma Zn²⁺ levels were 40% lower in mice placed on a ZnD diet (1.29 µM/g body mass). For ZnD mice returned to the ZnA diet (ZnR) for 2 wk, plasma Zn²⁺ levels (2.44 µM/g body mass) increased and were comparable to mice maintained on the ZnA diet.

The impact of dietary Zn²⁺ restriction and repletion on urinary Zn²⁺ excretion was also examined (Fig. 2B). Similar to plasma Zn²⁺, urinary Zn²⁺ levels were reduced in ZnD mice (3.43 ± 0.19 vs. 5.76 ± 0.84 µmol/day). However, after 2 wk of Zn²⁺ repletion, urinary Zn²⁺ levels remained low (3.27 ± 0.34 µmol/day) and were comparable to mice maintained on the ZnD diet. Collectively, these findings demonstrate that chronic dietary Zn²⁺ restriction is effective at inducing Zn²⁺ deficiency. Furthermore, while acute dietary Zn²⁺ repletion improves circulating Zn²⁺ levels, urinary Zn²⁺ excretion is not yet restored.

Limitations

This protocol was developed and optimized in adult, male, wild-type mice on a C57Bl/6 background. Various factors influence the handling of Zn²⁺, including genetic composition, age, and sex. Given the potential diversity in how Zn²⁺ is handled, the impact of dietary Zn²⁺ depletion and repletion may vary between experimental animals within a study. Consequently, the intricate interplay of genetic factors, age-related dynamics, and sex-specific differences can contribute to distinct responses to Zn²⁺ manipulation. This important consideration underscores the significance of establishing baseline Zn²⁺ levels. By obtaining a reference point, Zn²⁺ changes can be assessed not only between different experimental groups but also longitudinally, with each animal also serving as its own control. This rigorous approach provides a comprehensive assessment of the impact of Zn²⁺ dynamics on physiological and pathophysiological processes.

Troubleshooting

Problem 1: No changes in plasma and/or urinary Zn²⁺ levels after Zn²⁺ depletion.

Potential solution: Extend duration of Zn²⁺ Depletion Period

• To maintain whole-body Zn²⁺ homeostasis, Zn²⁺ is released from Zn²⁺ stores into the circulation. As such, a longer depletion period may be required to deplete Zn²⁺ stores and subsequently reduce serum and urine Zn²⁺ levels.

Problem 2: Reduced plasma and urinary Zn²⁺ levels after Zn²⁺ repletion.

Potential solution: extend the duration of Zn²⁺ repletion period.

Although ZnR mice (2.44 µM/g body mass) have increased plasma Zn²⁺ levels compared with ZnD mice (1.29 µM/g body mass), these levels are not yet comparable to ZnA mice (3.19 µM/g body mass). Furthermore, urinary Zn²⁺ levels are still depleted (3.27 ± 0.34 vs. 5.76 ± 0.84 µmol/day). Repletion of Zn²⁺ stores within organs proceeds repletion of serum and urinary Zn²⁺ levels. As such, a longer repletion period may be required when using serum and urine Zn²⁺ as markers of Zn²⁺ status.

DATA AVAILABILITY

This study did not generate/analyze (data sets/code). There are no newly generated materials associated with this protocol. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R21 DK119879-S (to A.C.U.) and R01 DK-133698 (to C.R.W.), the William Townsend Porter Predoctoral Fellowship from the American Physiological Society (to A.C.U.), American Heart Association Grant 16SDG27080009 (to C. R.W.), and American Society of Nephrology KidneyCure Transition to Independence grant (to C.R.W.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.-Y.W., K.E.M., K.M.E., and C.R.W. conceived and designed research; T.-Y.W., D.E., K.E.M., J.R., and C.R.W. performed experiments; T.-Y.W., D.E., K.E.M., J.R., K.H., and C.R.W. analyzed data; T.-Y.W., D.E., K.E.M., K.H., K.M.E., A.H., and C.R.W. interpreted results of experiments; T.-Y.W., K.E.M., J.R., K.H., and C.R.W. prepared figures; T.-Y.W., K.E.M., A.G.M., K.N., A.K., K.M.E., A.H., and C.R.W., drafted manuscript; T.-Y.W., K.E.M., A.C.U., A.G.M., K.N., A.K., K.M.E., A.H., and C.R.W. edited and revised manuscript; T.-Y.W., D.E., K.E.M., K.H., A.C.U., A.G.M., K.N., A.K., K.M.E., A.H., and C.R.W. approved final version of manuscript.