study B, which assessed the effect of mucin on heme iron bioavailability, the .... prohibited from eating until 4 h after ingestion of the doses. The amount of protein ...
Biol Trace Elem Res DOI 10.1007/s12011-012-9483-9
Effect of Trypsin and Mucin on Heme Iron Bioavailability in Humans Gustavo Cediel & Manuel Olivares & Diego Gaitán & Sebastian Flores & Alex Brito & Fernando Pizarro
Received: 3 May 2012 / Accepted: 27 July 2012 # Springer Science+Business Media, LLC 2012
Abstract In the human gastrointestinal tract, trypsin and mucin may affect the absorption of heme iron. However, these interactions have not been well-established. We determined the effect of trypsin and mucin on heme iron absorption in humans. Design: Twenty-eight apparently healthy females participated in two studies (14 per study). Study A evaluated the effect of trypsin on iron bioavailability. Subjects ingested 100 mg trypsin and 1.7 g mucin on 5 mg heme iron bioavailability on days 1, 2, 14, and 15, respectively. In study B, which assessed the effect of mucin on heme iron bioavailability, the subjects ingested hemin, hemin plus mucin, hemoglobin (Hb), and Hb plus mucin, on days 1, 2, 14, and 15, respectively. Results: In study A, the geometric means±1 SD of heme iron absorption were 5.1 % (3.1– 8.3), 2.9 % (1.6–5.1), 7.3 % (4.1–13.1), and 6 % (2.7–13) for hemin, hemin plus trypsin, Hb plus trypsin, and Hb plus mucin plus trypsin, respectively. In study B, the geometric means±1 SD of heme iron absorption were 16.4 % (10.5– 25.7), 13.1 % (9.0–18.9), 13.7 % (9.0–20.7), and 11.8 % (7.6–18.3) for hemin, hemin plus mucin, Hb, and Hb plus mucin, respectively. The ratio increased when Hb plus trypsin was ingested and decreased when hemin plus trypsin was ingested. There were no differences in other ratios with respect to the ratio on day 1 (P5 % on heme iron absorption (α0 0.05 and power080 %) by one-way ANOVA. Study Design In study A, the effect of 100 mg trypsin and 1.7 g mucin on 5 mg heme iron bioavailability was assessed. Both proteins were simultaneously incorporated to assess a potential synergic effect on heme iron bioavailability. The subjects ingested hemin, hemin plus trypsin, Hb plus trypsin, and Hb plus mucin plus trypsin on days 1, 2, 14, and 15, respectively. In study B, which assessed the effect of mucin on heme iron bioavailability, the subjects ingested hemin, hemin plus mucin, Hb, and Hb plus mucin, on days 1, 2, 14, and 15, respectively. The absorption of hemin on day 1 was used as a reference dose in order to compare studies. In both studies, labeled heme iron doses were administered; 37 kBq 59Fe being given on days 1 and 14 and 111 kBq 55Fe being given on days 2 and 15. Doses were administered after a nocturnal fast. Subjects were prohibited from eating until 4 h after ingestion of the doses. The amount of protein used in both studies was obtained from porcine (from a commercial provider, Sigma, St. Louis. USA). Iron isotopes of high specific activity were used as tracers (NEN, Life Science Products, Boston, MA). Doses of labeled heme were given to the subjects in gelatin capsules resistant to gastric digestion (number 0; Reutter, Santiago, Chile). Labeling of Hemin and Hb The labeled stroma-free Hb was prepared by using red blood cells from rabbits, based on the method described by Asenjo et al. [13]. Briefly, New Zealand rabbits, ∼3 kg in weight, received an intravenous injection of 74 MBq of 55Fe or 37 MBq
of 59Fe as ferric citrate (NEN, Life Science Products, Boston, MA) diluted in 10 mL of 0.16 mol NaCl/L. Fifteen days later, the rabbits were exsanguinated through cardiac puncture. The radioactive red blood cells were centrifuged (1,000×g for 15 min at 22 °C) and washed with saline then hemolyzed by freezing to obtain Hb. Finally, the Hb was dehydrated by lyophilization. Labeled freeze-dried Hb with a specific activity of 475 kBq of 59Fe and 2,460 kBq of 55Fe per milligram of heme iron was mixed in dry form with unlabeled Hb, resulting in a dose of 37 kBq of 59Fe or 111 kBq of 55Fe per administered dose. Radioactive hemin was prepared from labeled Hb as was previously described by Labbe and Nishida [14]. Then, the obtained hemin was washed with distilled water and dissolved in 0.1 N sodium hydroxide before administration. The compounds were packaged in gelatin capsules resistant to gastric digestion. Iron Status Venous blood samples were obtained on day 14 to determine iron status and on days 14 and 28 to measure circulating radioactivity in each experiment. Hb and mean corpuscular volume (MCV) were determined with a CELL-DYN 1700 instrument (Abbott Diagnostics, Abbott Park, IL). Free erythrocyte protoporphyrin (FEP) was determined with a hematofluorometer (ZP-M206D, AVIV Biomedical Inc., Lakewood, NJ). Serum ferritin (SF) was determined by enzymelinked immunosorbent assay [15], and transferrin saturation (Sat) was calculated as previously reported [16]. Anemia was defined as Hb 120 g/L, and at least two changes in other parameters (MCV 70 μg/dL red blood cell, and/or Sat
