Figure 1.
Sugar digests effects on iron-induced ferritin.
Measurement of Caco-2 cell ferritin formation from digests of Fe and solutions of sucrose (Sucrose+Fe) or glucose (Glucose+Fe) or fructose (Fructose+Fe) at an iron:sugar ratio of ≈ 1:2000. Equal amounts of iron (25 µg) were combined with sugar solutions (1.0 mL) and subjected to the Caco-2 in vitro digestion process. Digests with fructose alone and no added Fe (No Food Digest) were used as negative controls; digests with Fe alone (Fe) and Fe plus ascorbic acid (Fe + AA) were used as reference controls and positive controls, respectively. Values are means of data normalized to 10 ng of ferritin/mg protein in the reference control (Fe) ± SEM, n≥15. Based on an ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010).
Figure 2.
Effect of high-fructose corn syrup (HFCS) digests on iron-induced ferritin.
Measurement of Caco-2 cell ferritin formation from digests of Fe and fructose (Fructose+Fe), or Fe and HFCS-55 (HFCS+Fe) at an iron:fructose ratio of ≈ 1:2000. Equal amounts of iron (25 µg) were combined with fructose solutions (1.0 mL) and subjected to the Caco-2 in vitro digestion process. Digests with HFCS alone and no added Fe (No Food Digest) were used as negative controls; digests with Fe alone (Fe) and Fe plus ascorbic acid (Fe + AA) were used as reference controls and positive controls, respectively. Values are means of data normalized to 10 ng of ferritin/mg protein in the reference control (Fe) ± SEM, n ≥ 18. Based on an ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010).
Figure 3.
Effect of tannic acid (TA) and fructose, or TA and high-fructose corn syrup (HFCS), on iron-induced ferritin formation.
Measurement of Caco-2 cell ferritin formation from digests of Fe and fructose (Fructose+Fe), or HFCS-55 (HFCS+Fe), at an iron:fructose ratio of ≈ 1:2000, plus tannic acid at a 1∶1 molar ratio of Fe:TA. Equal amounts of iron (25 µg) were combined with sugar solutions (1.0 mL) and TA and subjected to the Caco-2 in vitro digestion process. Digests without TA are shown with lighter shading and digests with TA added are shown with darker shading. Values are means of data normalized to 10 ng of ferritin/mg protein in the reference control (Fe) ± SEM, n(Fe+AA+TA) = 4, n(TA alone) = 6, all other n = 18. Analysis of Figure 3A was based on a two-factor ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010). Analysis of Figure 3B was based on a one-factor ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010).
Figure 4.
Effect of phytic acid (PA) and fructose, or high-fructose corn syrup (HFCS), on iron-induced ferritin formation.
Measurement of Caco-2 cell ferritin formation from digests of Fe and fructose (Fructose+Fe), or HFCS-55 (HFCS+Fe), at an iron:fructose ratio of ≈ 1:2000, plus phytic acid at 1∶1, 1∶5 or 1∶10 Fe:PA molar ratios. Equal amounts of iron (25 µg) were combined with sugar solutions (1.0 mL) and PA and subjected to the Caco-2 in vitro digestion process. Digests of the above without the addition of PA are provided for reference. Digests with Fe alone (Fe), PA alone (PA Alone), and Fe plus PA (1Fe:10PA) were used as controls. Values are means of data normalized to 10 ng of ferritin/mg protein in the reference control (Fe) ± SEM, n(Fe+PA1:10) = 3, all other n ≥ 6. Analysis of Figure 4A was based on a two-factor ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010). Analysis of Figures 4B and 4C was based on a one-factor ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common within Figure 4B are significantly different (p ≤ 0.010) and similarly bar values with no letters in common within Figure 4C are significantly different (p ≤ 0.010).
Figure 5.
Carbohydrate effect on release of ferrozine-chelatable ferrous iron (Fe2+) in vitro.
50/L solutions of glucose, fructose, sucrose or mannitol were prepared with the addition of FeCl3 at a final concentration of 0.1 mmol/L. Solutions with iron alone and iron plus ascorbate were used as controls. Analysis for ferrozine-chelatable ferrous iron was performed after 2 hours incubation. Data in each column are presented as the mean ± SEM, n = 12 per group. Analysis was based on a one-factor ANOVA (p = 0.0001). Post-hoc analysis was done versus control. Fructose at a concentration of 50 mmol/L significantly increases ferrous iron levels in comparison to all other tested carbohydrate solutions; p < 0.0001, compared with 0.1 mmol/L FeCl3 alone.
Figure 6.
HepG2 iron-induced ferritin in response to carbohydrate treatments.
Measurement of HepG2 cell ferritin formation following treatment for 24 µmol/L ferric ammonium citrate (FAC) and one of the following: 15 mmol/L glucose (Glucose+FAC); 15 mmol/L glucose and 15 mmol/L fructose (Fructose+Glucose+FAC); 15 mmol/L fructose (Fructose+FAC). Cells treated with MEM alone (MEM), or fructose alone (Fructose Only), without the addition of FAC, were used as negative controls. Cells treated with 0.1 mmol/L ascorbate and 1 µmol/L FAC were used as positive controls. Values are means of data normalized to 400 ng of ferritin/mg protein in the reference control (MEM+FAC) ± SEM, n(MEM) = 4, n(Fructose Only) = 6, all other n ≥ 12. Based on an ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010).
Figure 7.
Fructose dose response on HepG2 cell iron-induced ferritin.
Measurement of HepG2 cell ferritin formation following treatment for 24 µmol/L FAC and one of the following: 1 mmol/L fructose (1 mM Fruc+FAC); 5 mmol/L fructose (5 mM Fruc + FAC); 15 mmol/L fructose (15 mM Fruc + FAC), to determine dose response of HepG2 cell ferritin relative to fructose concentration. Values are means of data normalized to 400 ng of ferritin/mg protein in the reference control (MEM+FAC) ± SEM, n ≥ 6. Based on an ANOVA (p<0.0001) with Tukey’s multiple comparisons test post-hoc analysis done on an all-pairwise basis, bar values with no letters in common are significantly different (p ≤ 0.010).