WO2015198337A1

WO2015198337A1 - Elisa for ferritin as marker for assessing bioavailability of iron using caco-2 cell line

Info

Publication number: WO2015198337A1
Application number: PCT/IN2014/000434
Authority: WO
Inventors: Madhavan Krishnapillai NAIR; Pullakhandam RAGHU; Punjal RAVINDER
Original assignee: Indian Council Of Medical Research
Priority date: 2013-06-28
Filing date: 2014-06-30
Publication date: 2015-12-30
Also published as: IN2013DE01927A

Abstract

This invention relates to a process for the assessment of bioavailability of iron in test foods comprising the steps of culturing Caco-2 cells in 6-well plate in a medium to obtain differentiated Caco-2 cells, subjecting test foods by simulated in vitro gastric digestion followed by in vitro intestinal digestion to obtain an intestinal digest, replacing the medium in the transwell plates with fresh medium adding the intestinal digest in an insert provided with a dialysis membrane and inserting the insert into the wells of the 6-well plates, subjecting the plate to incubation followed by removal of the digest and a step of further incubation, harvesting the cells, lysing the cells to obtain a cell lysate, followed by estimating cellular ferritin content in the cell lysate using a ELISA kit.

Description

IN/PA-211 1

FIELD OF THE INVENTION:

ί This invention relates to a process for assessing iron bioavailability in fortified foods, pharmaceuticals, agriculture biotechnological products.

This invention further relates to a process for measurement of iron bioavailability using Caco-2 cell line, derived from human colon adenocarcinoma.

BACKGROUND OF THE INVENTION:

Anemia is the most common and widespread nutritional disorder in the world. Globally an estimated 2 billion people-over 30% of the world's population are anemic. Most National surveys carried out in India show that the prevalence of anemia is over 60% among children and women in India (NHFS-3, 2006). Lack of iron and its bioavailability from habitual diets of Indians are considered to be the most important factors (Nair et , al., 2009). Apart from dietary factors the oxidation state of iron also influences the iron bioavailability. Food fortification is an integral part of measures to control iron- deficiency. Screening tools are needed for assessing bioavailability of iron from fortified foods, beverages, bio fortified crops or premixes and to select the best foods that provide highest iron bioavailability.

Iron bioavailability can be tested either in animal models or in human using radioactive or stable isotopic tracer methods (Bj5rn-Rasmussen et al., 1974 and Nair et al., 2013). In these methods, appearance of label in functional pool (hemoglobin) following the labeled test meal is tested. Although these methods are essential, associated radio-hazard cost of analysis with stable isotopic methods and inherent variation in iron bioavailability among subjects limit their use as a routine screening tool. IN/PA-211 2

This led to the development of simple in vitro models based on ionizable or dialyzable iron content after simulated in-vitro digestion of test food to assess the relative iron bioavailability of test foods (Rao et al., 1978, Miller et al., 1981). These methods have been used extensively, but suffer from the fact it has no living component involved.

Ferritin is a high molecular weight iron storage protein. It is chiefly found in liver, bone marrow and spleen and is inducible by iron. Ferritin levels in population can be an useful indicator to define the cause and stage of anemia. However, many of the available methods use radioisotopes of iron and no convenient and accurate methods are available in the art.

OBJECTS OF THE INVENTION:

It is therefore an object of this invention to propose a process for assessing bioavailability of iron, which is simple and convenient.

Another object of this invention is to propose a process for assessing bioavailability of iron, which is accurate.

Yet another object of this invention is to propose a process for assessing bioavailability of iron, which avoids the use of radio isotopes.

These and other objects and advantages of the invention will be apparent from the ensuing description, when read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Fig 1: Coupled in vitro digestion/Caco-2 cell ferritin induction model for assessing the iron bioavailability from test foods (Glahn et al., 1998) IN/PA-211 3

Figure 2: Effect of iron-source, ascorbic acid and phytic acid on ferritin induction in Caco-2 cells. Bars with different letters are significantly different (reproduced from Bejjani et al., 2007).

Figure 3: Ferritin induction in Caco 2 cells fed with chapattis made from wheat flour fortified with different iron salts at gastric pH 2 and 4.

Figure 4: Ferritin induction in Caco-2 cells from iron fortified rice compared to unfortified rice.

Figure 5: Iron bioavailability from fortified beverage premixes in Caco-2 cells (Raghu et al., 201 1)

DETAILED DESCRIPTION OF THE INVENTION:

Thus, according to this invention is provided a process for assessing iron bioavailability.

In accordance with this invention, the method comprises a coupled in vitro digestion/ Caco-2 cell model combined with cellular ferritin estimated by ELISA method.

Validation of ferritin as a marker of iron bioavailability in Caco-2 cells: The effect of iron salts in the presence and absence of iron adsorption inhibitor (phytic acid) and promoter (Ascorbic acid) has been evaluated in Caco-2 cells. The iron induced ferritin expression is measured using ELISA of ferritin. Phytic acid reduces while ascorbic acid enhances ferritin induction in Caco-2 cells, suggesting that measuring Caco-2 cells ferritin formation can be used as a surrogate marker of iron bioavailability in Caco-2 cells. IN/PA-211 4

Caco-2 cell ferritin induction with fortified rice and wheat flour;

Feeding foods prepared with fortified rice and wheat flour to Caco-2 cells ^~ induce ferritin. The method can be used for identifying best iron source for food fortification.

The method according to the invention is exclusively used for assessing the relative iron bioavailability from test foods compared to a reference standard of ferric chloride and ascorbic acid bioavailable.

Basic components of the tool include:

1. A 6-well plate with differentiated Caco-2 cells

2. Media

3. Transwell inserts fitted with a dialysis membrane

4. Lyophilized premixes for gastric and intestinal phases of digestion

5. Human ferritin ELISA kit

Food or meal is subjected to in vitro digestion with a simulated gastric and intestinal digestion conditions. The digest is then placed on to an upper chamber of a transwell plate and fed to the differentiated Caco-2 cells. The accessible iron is allowed to elicit a bio response as ferritin protein. The amount of ferritin formed in the cells is measured using an ELISA system. The induction of ferritin is directly proportional to the bioavailability of iron.

Fig. 1 shows the coupled in vitro digestion /Caco-2 cell ferritin induction model for assessing iron bioavailability of test foods.

The schematic diagram shows a single culture well (1) lined with Caco-2 cells. The insert (2) is provided with a dialysis membrane (3) and an insert ring (4). The methodology is depicted in Fig. 1 and essentially involves 5 steps. IN/PA-211 5

Step 1: Culture and differentiation of Caco-2 cells

Caco-2 cells obtained from the National Centre for Cell Sciences, Pune are being maintained in the laboratory.

Caco 2 cells are seeded at a density of 50,000 cells/ cm² in 6-well plates and grown in Dulbecco's modified Eagle's medium with 10% v/v fetal bovine serum, 25 mmol/L HEPES and 1% antibiotic antimycotic solution by incubating at 37°C in an incubator with a 5% CO₂/ 95% air atmosphere at constant humidity. The growth medium is changed every 2 days till 13 days to obtain differentiated Caco-2 cells and is used for iron bioavailability experiments.

Step 2: In vitro digestion of test food

The test foods (cereal based foods, premixes, liquid beverages etc) are subjected to simulated in vitro gastric and intestinal digestion as described below.

Gastric pepsin digestion:

Pepsin reagent: The reagents are prepared shortly before use. Dissolve- 0.2 g pepsin in 5 mL of 0. 1 mol/L HC1, and add 2.5 g of Chelex- 100 (to remove endogenous iron). Mix the contents on a tabletop shaker for 30 min. Filter it through a 1.6X20 cm filtration column and collect the pepsin solution. Add 5 mL of 0. 1 mol/L HC1 to the column and collect the filtrate into the pepsin solution. The final volume of the pepsin solution is about 8 mL.

Peptic digestion: Cooked food stuffs are used for bioavailability test. Homogenize the food sample. Take about 1 to 2 g and adjust the pH to 2.0 with 5.0 mol/L HC1. Transfer the sample to a 50-mL screw-cap culture tube, and add 0.5 mL of the pepsin solution per 10 mL of sample. IN/PA-211 6

Place this horizontally on a rocking shaker and mix the contenst at 55 oscillations/ min about for 60 min to obtain the sample digested at gastric condition.

Preparation of pancreatin/bile solution: Prepare 0.05 g pancreatin and 0.3 g bile extract in 25 mL of 0. 1 mol/L NaHC0₃. Add Chelex- 100

( 12.5 g) and mix it for 30 min on a tabletop shaker. Pour the mixture into a 1.6X 20 cm filtration column and collect the pancreatin/bile filtrate. Add 10 mL of 0. 1 mol/L aHCOa to the column and collect the filtrate into the pancreatin/bile solution. The final volume of the pancreatin/bile solution is about 27 mL.

Intestinal digestion: The pH of the sample digested at gastric condition , (referred to as the "digest") is raised to pH 6 by drop-wise addition of 1 mol/L NaHC03. Add 2.5 mL of pancreatin-bile extract mixture per 10 mL of the digest. The pH is adjusted to 7 with NaOH, and the volume brought to 15 mL with 120 mmol/L NaCl and 5 mmol/L KC1. Mix the intestinal digest thoroughly and place it in the apical chamber of the 6 well plates as described below.

Step 3: Preparation of the transwell plates and feeding the cells:

12 h prior to the experiment, the spent media from the 6 well plate ( 13 day post-seeding) is replaced with 1 mL of fresh Minimum Essential Medium (MEM) supplemented with 10 mmol/L PIPES (piperazine-N,N'- bis-[2-ethanesulfonic acid]), 1% antibiotic-antimycotic solution (Sigma), hydrocortisone (4 mg/L) , insulin (5 mg/L) , selenium (5 μg/L), triiodothyronine (34 g/L) and epidermal growth factor (20 g/L). A sterilized insert fitted with a dialysis membrane, is inserted into the wells as shown in Fig. 1. IN/PA-211 7

1.5-mL aliquot of the intestinal digest is taken and introduced it into the upper chamber of the insert. The plate is covered and incubated on the rocking shaker at 6 oscillations/ min for 120 min. At the end of this period, the insert ring is removed with the digest and 1 mL of MEM is added to each well and incubated in a C02 incubator for 22 h without shaking. The cells are harvested for analysis as described below.

Negative and positive controls: A negative control with 2 mL of saline and a positive control with 2 mL of ferric chloride/ ascorbate solution at 1 : 10 ratio of iron to ascorbic acid in saline are included instead of food sample and process as described above. Ensure minimal external iron contamination is ensured during digestion reaction.

Step 4: Harvesting cells

The Caco 2 cell monolayer is harvested for ferritin analysis. Remove the medium covering the cells and add 2 mL volume of a lOmmole/L phosphate buffer saline pH 7.2 (PBS). Discard the medium and scrape the cells in PBS. The contents are sonicated for a period of 2 min to lyse the cells.

The protein content of the lysate is estimated by micro BCA kit method against BSA standard. The cell lysates are stored at -20°C until ferritin analysis as described below.

Step 5: Estimation of cellular ferritin content using in-house ELISA kit

Ferritin in the cell lysate is estimated by an in- house ELISA kit. IN/PA-211 8

Coating of Plate: In-house antiserum (antiserum of human liver ferritin in rabbits) is diluted to 1:5000 times in 0.05M Carbonate buffer pH 9.6. 250 μΐ is added to each well of microtiter plate, covered with a plate sealer and incubate overnight at 4°C in a refrigerator.

Blocking: The plate is allowed to reach room temperature (RT). The contents are discarded and 250 μΐ of Carbonate buffer containing 0.5% BSA (assay buffer) is added. The plate is incubated at RT for 60min. The contents are discarded and washed as described below.

Washing: The plate is washed by either using a automatic plate washer OR manually.

For manual washing procedure, 300 μΐ of 0. 15M Phosphate buffered Saline pH 7.2 containing 0.05% Tween- 20 (PBST) is added. Incubated for 3 min in a rocking shaker and discarded the contents. Repeat the above process two more times.

Standard ferritin and sample: Stock ferritin standard is diluted in PBST containing 0.5% BSA (assay buffer) to obtain 5, 10,25,50 100 and 200 ng/ml. The cell lysates are either used as such or diluted 5times in assay buffer for the assay.

Assay: Add 190μ1 of assay buffer to the designated wells and 200 μΐ in blank. ΙΟμΙ of standard / sample as added in designated wells and incubated at RT for 2 hours. The plate is washed as mentioned above. 200 μΐ conjugate (antiferritin antibody conjugated to HRPO) diluted to 1 :6000 times in assay buffer is added and incubated for 2 hours at RT. I /PA-211 9

The plate is washed as mentioned above. 200 μΐ of substrate (Hydrogen Peroxide and orthophenylene diamine dihydrochloride (OPDD) in Phosphate-citrate buffer pH 5) is added to each well and incubated for 30 min. The color intensity is measured at 492nm in an ELISA plate reader.

Calculation: The net OD obtained after subtracting blank OD from standards OD and is plotted on log-log sheet against concentration of standard. From the net OD of sample ferritin in the given sample is calculated.

The invention will now be explained in greater detail with the help of the following non-limiting examples.

Examples

The specific examples where the above methodology applied are given below.

The test involved assessing induction of Caco-2 cell ferritin as a surrogate of iron bioavailability in humans from various sources of iron in the presence and absence of ascorbic acid (an enhancer of iron absorption) and phytic acid (inhibitor of iron absorption) .

Example 1: Iron bioavailability from plants sources: pea ferritin as a source of iron:

The above method was used for assessing iron bioavailability from pea ferritin. Figure -2 shows the induction of ferritin in Caco-2 cells fed iron from FeSO₄ and plant source pea ferritin in the presence and absence of phytic acid and ascorbic acid. ΙΝ/ΡΑ-2Π 10

The results demonstrate that Caco-2 cells fed with FeS0₄ and pea ferritin induced ferritin more than the control. Induction of ferritin from both the sources was many folds higher in the presence compared to the absence of ascorbic acid. Ferritin induction with pea ferritin and phytic acid was similar to the control.

The direction and magnitude of changes in ferritin induction in Caco 2 cells in the presence and absence of ascorbic acid and phytic acid from iron salt and plant ferritin confirms that the cellular ferritin induction in Caco-2 cells can predict the iron bioavailability in humans.

Example 2: Iron bioavailability from iron fortified wheat flour:

Ferritin induction in Caco-2 cells as a surrogate of iron bioavailability from Indian bread (chapatti) was assessed using the method described above. The experiment was also carried out at pH 2 and 4 to assess the effect of gastric pH on iron bioavailability.

Whole wheat flour was fortified with different iron salts at 60mg/kg level.

Chapattis were made. These were fed to the Caco-2 cell system after in vitro digestion and the results are given in Figure -3.

The results suggest that among all the iron salts tested the ferritin induction was greatest with NaFeEDTA, which is in agreement with several animal and human studies in a phytate rich food matrix such as whole wheat flour (Muthayya et al, 2012). There was no difference in induction of ferritin when food was digested at gastric pH 2 and 4.

Example 3: Iron bioavailability from iron fortified rice:

Ferritin induction in Caco-2 cells as a surrogate of iron bioavailability was tested with fortified rice. IN/PA-211 1 1

Induction of ferritin in Caco-2 cells was greater with fortified rice compared to unfortified rice (Figure-4). Efficacy trial carried out in children fed with iron fortified rice as part of MDM reported that iron fortified rice is effective in improving iron stores (serum ferritin) compared to unfortified rice (Radhika et al., 201 1)

Example 4: Ferritin induction in Caco-2 cells from iron fortified beverage premixes:

Iron bioavailability from iron fortified beverage premixes in the presence of wheat flour based Indian bread was tested using the above method.

Two combinations of micronutrient premixes were tested. Wheat flour chapattis were mixed with Premix - 1 contained iron, zinc and vitamin A and premix- 2 iron, zinc, vitamin A and Vitamin C and fed to Caco-2 cells for ferritin induction (Figure 5). Induction of ferritin was minimal with food, but was markedly higher in the presence of ascorbic acid (premix - 2), which is in accordance with the enhancing effect of vitamin C on iron absorption.

The method can be used in the following fields: Food industry:

❖ Screening of various fortified foods such as infant formula, breakfast cereals and dairy products

❖ Screening for bioavailable sources of iron for food fortification (premixes)

Pharmaceuticals:

❖ Can be used for assessing bioavailability iron from tablets, syrups etc.

❖ Assessing efficacy of pre and probiotics on iron bioavailability IN/PA-211 12

❖ Screening of bioactive phytochemicals on iron bioavailability

Agriculture biotechnology and Nutritional sciences:

❖ Screening germplasm for selecting breeder lines for bioavailable iron Ferritin ELISA Diagnostic Kit

❖ Assessment of iron status-clinical diagnosis and management of true iron deficiency

❖ Evaluation of iron overload

❖ For management of patients suffering from β-thalssemia.

Claims

IN/PA-211 13 WE CLAIM:

1. A process for the assessment of bioavailability of iron in test foods comprising the steps of culturing Caco-2 cells in 6-well plate in a medium to obtain differentiated Caco-2 cells,

subjecting test foods by simulated in vitro gastric digestion followed by in vitro intestinal digestion to obtain an intestinal digest,

replacing the medium in the transwell plates with fresh medium adding the intestinal digest in an insert provided with a dialysis membrane and inserting the insert into the wells of the 6-well plates,

subjecting the plate to incubation followed by removal of the digest and a step of further incubation, harvesting the cells, lysing the cells to obtain a cell lysate, followed by estimating cellular ferritin content in the cell lysate using a ELISA kit.

2. The process as claimed in claim 1, wherein said steps of gastric digestion and intestinal digestion are carried out on test foods selected from cereal based foods, premixes, liquid beverages.

3. The process as claimed in claim 1 , wherein said step of gastric digestion is carried out by homogenizing the food sample followed by addition of pepsin solution thereto followed by thorough mixing for about 60 min to obtain the gastric digest.

4. The process as claimed in preceding claims, wherein said step of intestinal digestion is carried out by adding a base to gastric digest to raise the pH to 6, followed by adding a pancreatin-bile extract mixture, raising the pH to 7 by addition of an alkali, followed by addition of salts thereto and mixing to obtain the intestinal digest.

5. The process as claimed in claim 4, wherein "said base is sodium bicarbonate. IN/PA-211 14

6. The process as claimed in claim 4, wherein said alkali is sodium hydroxide.

7. The process as claimed in claim 4, wherein said salts are selected from sodium chloride, potassium chloride.

8. The process as claimed in claim 1 , wherein said medium is Eagle's medium with fetal bovine serum, HEPES and antibiotic antimycotin solution.

9. The process as claimed in claim 1 , wherein said fresh medium comprises Minimum Essential Medium (MEM) supplemented with piperazine-N,N'-bis-[2-ethanesulfonic acid (PIPES), antibiotic- an timycotic solution, hydrocortisone, insulin, selenium, triiodothryonine and epidermal growth factor.

10. The process as claimed in claim 1 , wherein said step of incubation is effected for 120 minutes with shaking.

1 1. The process as claimed in claim 1, wherein said step of further incubation is effected for 22 hours without shaking.

12. The process as claimed in claim 1 , wherein the harvested cells are lysed by sonication.

13. The process as claimed in claim 1 , wherein for the estimation of cellular ferritin content, the wells of the microtiter plates of the

ELISA kit are coated with an in-house antiserum, and cell lysate IN/PA-211 15 is added designated wells and standard ferritin in designated

wells,

a conjugate is added to the wells and incubated,

followed by addition of hydrogen peroxide and orthopheylene

diamine dihydrochloride (OPDD) and incubation for the

development of a colour,

measuring the intensity of the colour as optical density, followed by calculation of the ferritin content therefrom.

14. The process as claimed in claim 13, wherein said in-house antiserum is antiserum of human liver ferritin in rabbits.

15. The process as claimed in claim 13, wherein said conjugate is an antiferritin antibody conjugated to HRPO.