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Human Nutrition and Metabolism

A Low-Molecular-Weight Factor in Human Milk Whey Promotes Iron Uptake by Caco-2 Cells¹

Department of Food Science and ^* U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, NY 14853

²To whom correspondence should be addressed. E-mail: rpg3{at}cornell.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The iron bioavailability of human milk (HM) is substantially greater than that of cow’s milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 µmol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48%. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60%, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (<10 kDa) in human milk enhances iron absorption.

KEY WORDS: • iron bioavailability • Caco-2 cells • human milk • cow’s milk • whey

The bioavailability to human infants of iron from human milk (HM)² is greater than that from cow’s milk (CM) on a fractional basis. The factors responsible for the greater Fe bioavailability of HM compared with CM have not been identified. However, HM contains lower concentrations of casein, phosphates and calcium, factors thought to inhibit Fe absorption, and higher concentrations of absorption-enhancing components such as lactoferrin, ascorbic acid, lactose, cysteine, taurine and inosine (1). Of these enhancing factors, HM lactoferrin has received the most attention.

The interest in lactoferrin began in 1939 when it was first identified as a "red protein" in bovine milk. It was not until 20 y later that the purified protein was isolated from milk (2). Because Fe from HM is highly bioavailable and because HM contains high concentrations of lactoferrin, researchers speculated that this Fe-binding protein was responsible for making the metal readily absorbable. This was particularly interesting in the pediatric arena, because HM often represents the first and sole food for infants, at least for the first six months of life. It could be possible, researchers argued, that lactoferrin, with its bound Fe, was reaching the brush-border surface of the intestinal mucosal cells of the infant intact, due to the very immature GI system in the infant (low levels of peptic enzymes, high gastric pH and low levels of intestinal enzymes), and that the iron-lactoferrin complex was absorbed intact.

However, more recent reports began to cast doubt on the importance of lactoferin as a promoter of Fe absorption (3–5). Interest in lactoferrin and its effect on Fe absorption has waned in recent years. A literature search on lactoferrin documented increasing interest in its bacteriostatic, fungicidal, antioxidant and antiviral effects, some of which depend on the fact that the protein can sequester Fe from the environment. To date, the Fe uptake–enhancing component in HM, the so-called milk factor, has not been found.

In this study, we determined the Fe bioavailability of HM and CM using the Caco-2 cell model system. Both HM and CM were first fractionated into fat, casein, and whey. These fractions were subsequently replaced with media to obtain modified milks, i.e., milks lacking fat, casein, or whey. The Caco-2 cell uptake of Fe from these modified milks was compared to that from intact milk. An important and unique feature of this study design was that removing one specific fraction or component from the milk made it possible to identify which components inhibit or enhance Fe uptake. To our knowledge, there are no published studies that measure the Fe bioavailability of milk in this way. Therefore, the specific objectives of this study were to determine whether a milk factor indeed exists, and if so, to identify the HM fraction that contains the Fe uptake–enhancing component.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Glassware. All glassware was rinsed with 10% HCl and 18 M H₂O before use in order to avoid mineral contamination.

    Cell cultures. The Caco-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Stock cultures were maintained in DMEM (GIBCO, Grand Island, NY), pH 7.4, supplemented with 10% (v/v) fetal bovine serum (GIBCO), 25 mmol/L HEPES and 1% antibiotic-antimycotic solution (GIBCO). Cells were cultured at 37°C in an incubator with an atmosphere of 5% CO₂ and 95% air at constant humidity. The medium was replaced every 2 d. For uptake experiments, cells (passages 30 to 35) were grown in collagen-treated 6-well or 24-well plates at an initial seeding of 50,000 cells/cm². Uptake experiments were conducted at 14 d postseeding.

    HM and CM fraction preparation. The HM was obtained from the USDA Agriculture Research Service (USDA/ARS) Children’s Nutrition Research Center (CNRC; Houston, TX). The CNRC obtained informed written consent from each mother donating the milk. The HM was fractionated as described by Fransson and Lönnerdal (6). To obtain HM - fat, HM - casein and HM - whey, HM was first ultracentrifuged at 150,000 x g for 1 h at 4°C in an Optima XL-100K ultracentrifuge (Beckman Instruments, Palo Alto, CA). The resulting components were an upper phase (fat), a supernatant consisting of whey (also known as the serum phase), and a pellet containing the casein. The fat was removed with a spatula into a sterile preweighed test tube and replaced with an equal weight of minimum essential medium (MEM; GIBCO). The resulting product was labeled HM - fat.

To prepare HM - casein, the fat and whey components were transferred to a clean test tube. The casein pellet was saved in a preweighed tube. An amount of MEM equal to the weight of the casein was then added to the fat and whey components. To prepare HM - whey, the casein and fat components were combined, and MEM was added to replace the whey (Fig. 1).

View larger version (35K): [in this window] [in a new window]   FIGURE 1 Fractionation procedure for human milk (HM). The same procedure was applied to cow’s milk (CM); MEM = minimum essential medium. Ultrafiltration of the whey yielded a 10-kDa retentate (10kR; particles > 10 kDa) and filtrate (10kF; particles < 10 kDa).

The same fractionation method was used to prepare CM - fat, CM - casein and CM - whey. The CM used was pasteurized, vitamin D–fortified whole milk purchased at the local Cornell Dairy Store (Ithaca, NY).

    Ultrafiltration of whey. The HM and CM whey components obtained by ultracentrifugation were ultrafiltered using an Amicon 8200 stirred ultrafiltration cell with a 10-kDa membrane disc (Millipore, Bedford, MA) at an operating pressure of 276 kPa. This produced a 10-kDa retentate (10kR; components > 10 kDa) and filtrate (10kF; components < 10 kDa) (Fig. 1). To prepare HM - 10kF, HM - 10kR, CM - 10kF and CM - 10kR whey, intact milk samples were ultracentrifuged as described above. Fat and casein were then transferred to a clean test tube. The whey components were ultrafiltered as described above, and the 10kF or 10kR whey fractions were added to the appropriate samples (i.e., the 10kR fraction was added to the HM or CM - 10kF whey and the 10kF fraction was added to the HM or CM - 10kR whey; Fig. 1). The weight of the component that was not included was replaced with MEM.

    Effect of the HM fractions on Caco-2 cell Fe uptake. To study the effects of HM, HM - fat, HM - casein and HM - whey on Fe uptake, 1 mL of each sample was combined with 14 mL of MEM. This dilution was necessary because a more concentrated milk sample proved to be lethal to the cells. A 24-well plate was prepared with 0.5 mL of MEM. The following day, 0.75 mL of each prepared HM sample was transferred to a microcentrifuge tube. Iron (as ferrous sulfate) was added to some samples to bring the final concentration to 10 or 20 µmol/L Fe. The MEM on top of the cells was aspirated. After vortexing the contents of each microcentrifuge tube, 0.5 mL of this solution was placed on top of the cells. Each sample was prepared in duplicate for each 24-well plate. Five plates were prepared, all on the same day. Each fractionation process was performed separately and independently.

After incubating for 24 h to allow ferritin formation, the contents of each well were aspirated, and the cells were washed twice with saline. Cells were then sonicated for 15 min at 4°C in the presence of 0.5 mL of 18 M H₂O in an Elma Transsonic Digital sonicator (Lab-Line Instruments, Melrose Park, IL). The wells were scraped with a pipette tip. The contents of the wells were transferred to microcentrifuge tubes and stored at -20°C until analysis for cell protein and ferritin.

    Effect of the 10kF and 10kR whey fractions on Fe uptake. The effects of the HM and CM 10kF and 10kR whey fractions on Fe uptake were compared. Approximately 1 g of each 10kF and 10kR whey fraction was dissolved in 14 mL of MEM, and 0.75 mL of each sample was then transferred to a microcentrifuge tube. Because the ferritin levels of cells treated with the HM fractions did not differ from those of the control cells, Fe (as ferrous sulfate) was added to the samples to achieve a final concentration of 10 µmol/L Fe in each well. After 24 h the cells were rinsed, sonicated, transferred to microcentrifuge tubes and stored at -20°C until analysis for cell protein and ferritin. Each sample was prepared in duplicate for each 24-well plate, and there were five independent replications.

    Effect of the HM and CM fractions on Caco-2 cell Fe uptake after in vitro digestion. To study the effect of enzymatic digestion of the HM fractions on Fe uptake, all HM fractions (HM, HM - fat, HM - casein, HM - whey, HM - 10kF whey, HM - 10kR whey) and the corresponding CM fractions were subjected to in vitro digestion. Details of the procedure were as previously described (7). To 2 mL of each sample, 3 mL of 140 mmol/L NaCl, 5 mmol/L KCl was added. After peptic digestion the volume of each sample was brought to 10 mL (instead of 15 mL) with 140 mmol/L NaCl, 5 mmol/L KCl. Iron (10 µmol/L as ferrous sulfate) was added to each sample. Peptic digestion was carried out at pH 4 rather than pH 2 to simulate gastric pH conditions in infants. All samples were prepared on the same day, using cells grown on 6-well plates, and there were five independent replications.

    Chemical analyses. Cell and sample protein were assessed using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA), based on the Lowry assay. Mineral concentration was measured via inductively coupled argon plasma emission spectrometry (ICAP Model 61E Trace Analyzer; Thermo Jarrell Ash, Franklin, MA). Caco-2 cell ferritin was assessed using a one-stage sandwich immunoradiometric assay (FER-IRON II Ferritin Assay; RAMCO Laboratories, Houston, TX).

    Statistical analysis. Data were analyzed by one-way ANOVA after testing for normality and equal variance with Prism software (GraphPad Software, San Diego, CA). Samples with unequal variances were logarithmically transformed and analyzed by one-way ANOVA. Selected pairs of means were compared by Bonferroni posttest. Values of P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Milk composition. According to Geigy Scientific Tables (8), mature HM contains 13 to 83 g fat/kg and 1.4 to 6.8 g casein/kg; CM contains 34 to 61 g fat/kg and 23 to 28 g casein/kg. The high CM casein concentration found in this study (Table 1) could be attributed to other components that precipitated with the casein following centrifugation, including enzymes, entrapped minerals, salts, cells, leukocytes, water and so forth.

    Effect of the HM fractions on Caco-2 cell Fe uptake. The ferritin/cell protein ratios obtained with the fractions with no added Fe were very close to the basal ferritin/cell protein ratios obtained from Caco-2 cells that were not treated with any sample. This suggests that the endogenous Fe concentration of these fractions was too low to elicit any change in cell ferritin formation. Adding 10 and 20 µmol/L Fe dramatically increased cell ferritin formation (Fig. 2).

View larger version (44K): [in this window] [in a new window]   FIGURE 2 Ferritin levels of Caco-2 cells treated with human milk (HM) fractions with 0, 10 and 20 µmol/L Fe added as FeSO4. Bars are means ± SEM, n = 5. Means for each iron concentration (0, 10 and 20 µmol/L) without a common letter differ; P < 0.05.

    Effect of the 10kF and 10kR whey fractions on Fe uptake. Both the CM and HM 10kF whey fractions caused greater ferritin formation than the 10kR whey fractions (Fig. 3). Comparing the 10kF whey components, the CM whey caused markedly greater ferritin formation, whereas the HM whey caused greater ferritin formation among the 10kR whey components.

View larger version (32K): [in this window] [in a new window]   FIGURE 3 Ferritin levels of Caco-2 cells treated with human milk (HM) and cow’s milk (CM) 10-kDa filtrate (10kF; components < 10 kDa) and retentate (10kR; components > 10 kDa) whey fractions with added iron (10 µmol/L as FeSO4). Bars are means ± SEM, n = 5. Means without a common letter differ; P < 0.05.

View larger version (41K): [in this window] [in a new window]   FIGURE 4 Ferritin levels of Caco-2 cells treated with digested human milk (HM) and cow’s milk (CM) fractions. All test fractions were supplemented with iron (10 µmol/L as FeSO4) and subjected to in vitro digestion/Caco-2 cell model. Bars are means ± SEM, n = 5. Means without a common letter differ; P < 0.05.

The HM - 10kF whey fraction elicited significantly lower (-60%) ferritin formation than the HM fraction, but did not differ from the HM - whey fraction (Fig. 5). The HM - 10kR whey, CM - whey and CM - 10kF whey fractions did not affect cell ferritin formation (Fig. 5).

View larger version (40K): [in this window] [in a new window]   FIGURE 5 Ferritin levels of Caco-2 cells treated with digested human milk (HM) and cow’s milk (CM) whey fractions, including 10-kDa filtrate (10kF; components < 10 kDa) and retentate (10kR; components > 10 kDa) fractions. All test fractions were supplemented with iron (10 µmol/L as FeSO4) and subjected to in vitro digestion/Caco-2 cell model. Bars are means ± SEM, n = 5. Means without a common letter differ; P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study shows that it is the low-molecular-weight components of whey that enhance Fe uptake. Removing the 10kF whey fraction had the same effect as removing the total whey component from HM. Conversely, removing the 10kR whey fraction did not affect ferritin formation. Thus, the decrease in ferritin formation due to removing whey can be attributed to the low-molecular-weight whey components, not to the high-molecular-weight whey components.

The decrease in ferritin formation when whey was removed was evident only when the samples were digested, i.e., when pepsin, pancreatin and bile were added to the samples before treating the cells. However, removing the whey from HM did not affect iron bioavailability when the samples were not digested (Fig. 2).

The decrease in cell ferritin levels caused by digested HM - whey but not undigested HM - whey suggests that the Fe added to intact milk might form a complex with a whey component, rendering it unavailable to the cells without enzymatic digestion. The enzymatic digestion may also lead to the digestion of an Fe uptake inhibitor, thus increasing the bioavailability of Fe. Alternatively, it could also form a digestion product that promotes bioavailability by chelating Fe and keeping it soluble.

The fact that the 10kF whey fraction (<10 kDa) had the greatest Fe uptake–enhancing effect compared to the 10kR whey fraction (>10 kDa) rules out the possibility that lactoferrin, an 80-kDa glycoprotein (10), is the only or main Fe uptake–enhancing component. After ultrafiltration, lactoferrin and the other whey proteins [-lactalbumin (14.2 kDa), serum albumin (68 kDa) and immunoglobulins (150 to 900 kDa)] would be retained in the 10kR fraction. The results of this study are in agreement with the observations of Hussain et al. (11) who found that it was the <10-kDa fraction of HM whey that led to higher iron transfer and uptake in Caco-2 cells grown in bicameral chambers. Likewise, Serfass and Reddy (12) recently found that low-molecular-weight fractions derived from a 10-kDa filtrate were as effective as ascorbate and nitrilotriacetate in solubilizing Fe at neutral pH. However, when they pretreated the low-molecular-weight whey component with pepsin, pancreatin and bile extract and added extrinsic Fe, the amount of soluble Fe did not differ from that found with nondigested low-molecular-weight whey (12).

The HM - fat fraction elicited the greatest cell ferritin formation, suggesting that it is the fat component of HM that inhibits Fe uptake (Figs. 2and 4). It is possible that Fe added to intact HM is adsorbed to the fat globule membranes (composed of proteins and phospholipids). It is also possible that Fe binds to free fatty acids or fatty acids released from lipolysis of triacylglycerols (by human milk lipase) or to phospholipids, forming insoluble Fe soaps. Iron bound to the fat globule membranes or to fatty acids is not likely to be absorbed by the cells. The present study shows that HM fat reduces available iron whether digested (Fig. 4) or undigested (Fig. 2).

Removing the casein from HM did not affect cell ferritin formation regardless of whether the samples were digested. Thus, HM casein did not affect iron uptake. Human milk casein, which is composed largely of ß-casein with small amounts of -casein and very little to no -casein (9,13), represents a small fraction of the total protein component of HM. The casein fraction of HM total protein can be as low as 20% in early milk to 40% in more mature milk (9), and the percentage of casein in HM ranges from 0.14 to 0.68% by weight (8). In the present study, the calculated percentage of casein in HM was 0.7% (Table 1). Because this is such a small fraction, the amount of Fe binding to casein is probably low, which could partly explain the lack of effect on Fe uptake. It is also possible that the casein is already saturated with other minerals, such as calcium, phosphorus, zinc, iron, magnesium and copper, preventing the added Fe from binding.

Results for the CM - casein, CM - fat and CM - whey fractions differed from those obtained with the HM fractions. The CM - casein fraction elicited the greatest ferritin formation in the cells, which suggests that it is the casein component in CM that acts as an Fe uptake inhibitor, as opposed to the fat component in HM. Bovine casein, which is composed of , ß and caseins, has been shown to be an Fe uptake inhibitor, both in vivo (14) and in vitro (15,16). The low bioavailability of Fe from CM casein might be explained by 1) low dialyzability of Fe, as observed in infant formulas that contain casein as the main protein fraction (17) and in commercial casein (16); 2) very slow enzymatic digestion by the brush-border enzyme alkaline phosphatase, which liberates the phosphorus-Fe complex from the casein (18) and 3) very strong affinity of the casein for Fe, which prevents it from interacting with the intestinal cell receptor.

However, removing fat from CM did not affect ferritin formation. King et al. (19) studied the distribution of natural versus added ⁵⁹Fe isotopes in whole, unpasteurized and unhomogenized CM. Natural ⁵⁹Fe was incorporated into milk by administering the isotope via the subgular vein of the cow (intrinsic labeling), whereas the added ⁵⁹Fe was added directly to freshly drawn milk (extrinsic labeling). The milk was then fractionated. The researchers found that most of the added ⁵⁹Fe radioactivity (98%) was associated with the skim milk protein (i.e., devoid of fat). However, natural ⁵⁹Fe was highly concentrated at the fat globule membrane. Although the authors failed to explain the differences in distribution between the intrinsic and extrinsic isotope, this type of study might explain why removing fat from whole CM milk has no effect on Fe uptake. When Fe was added to whole CM, it did not bind to the fat.

Removing whey from CM did not affect ferritin formation in the present study, nor did removing the 10kF whey. Although -lactalbumin and lactoferrin are the main whey proteins in HM, ß-lactoglobulin is the main whey protein in CM, followed by -lactalbumin and proteose-peptones, immunoglobulins and serum albumin (20).

The 10kF CM whey fraction (<10 kDa) elicited the greatest Fe uptake in Caco-2 cells (Fig. 3). It is important to note that the whey fractions presented here were added to the cells on an equal-weight basis and that the samples were not digested. The weights in no way reflect the proportions found in either HM or CM. On an equal-weight basis, the 10kF CM whey elicited the greatest ferritin formation, whereas the higher molecular weight CM whey elicited the least ferritin formation (Fig. 3). Also, the 10kF whey fractions that contained the lowest concentrations of protein (0.4 and 4.5 g/L) elicited the greatest Fe uptake, and the 10kR whey fractions that contained the highest concentrations of protein (98.3 and 40.3 g/L) elicited the least Fe uptake. The lower Fe uptake elicited by the 10kR whey could be due to protein aggregation that makes the Fe unavailable for cell uptake. The lower protein concentration of the 10kF whey suggests that the Fe uptake–enhancing component in this fraction might be a nonprotein component. However, it could be a compound bound to a peptide or protein that can enhance Fe uptake when pepsin, pancreatin and bile are added, as previously shown.

In conclusion, there seems to be an Fe uptake–enhancing milk factor in HM, which is found in the whey. More specifically, it is the low-molecular-weight fraction (<10 kDa) that promotes Fe uptake. The high-molecular-weight fraction of whey, which includes lactoferrin among other whey proteins and components, is not the Fe uptake–enhancing fraction.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 03, April 2003, San Diego, CA [Etcheverry, P., Miller, D. D. & Glahn, R. P. (2003) Human milk fractionation and its effect on iron uptake by Caco-2 cells. FASEB J. 17: LB365 (abstract)].

³ Abbreviations used: CM, cow’s milk; HM, human milk; MEM, minimum essential medium; 10kF, 10-kDa filtrate; 10kR, 10-kDa retentate.

Manuscript received 29 July 2003. Initial review completed 26 August 2003. Revision accepted 30 September 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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