There are no reference materials that are certified for heme iron. The accuracy of the heme iron analysis was validated by demonstrating that the material balance (100% × (heme iron + non-heme iron)/total iron) for the in-house control samples and selected meat samples were close to 100%.
Initial results in the development phase gave poor recoveries. It was determined that traditional molecular absorbance measurements of heme iron (following acidified extraction) were biased high due to background absorbance. This problem was reduced by using measurements at 500 nm and 600 nm to extrapolate to the background at 540 nm. However, these values were still greater than those obtained by FAAS and gave material balances in excess of 100%. The validity of the acidified acetone extraction was confirmed by HPLC. Sample extracts provided single peaks whose retention times agreed with the hemin standard and whose quantity agreed with the FAAS analysis (data not shown). Consequently, FAAS was used for all the heme iron values reported here.
Measurements of total iron and non-heme iron by FAAS (following acid digestion) were biased low due to an undetermined interference. Analysis of the digested samples using FAAS and the method of additions confirmed suppression of the iron signals by 20% to 40% (data not shown). Accurate analysis of the acid digests was obtained using ICP-AES, yielding values equivalent to those obtained using FAAS and the method of additions. Thus, material balances of approximately 100% were achieved by analyzing heme iron by FAAS and total iron and non-heme iron by ICP-AES.
In general, the values for total iron and heme iron (Tables 2-4) determined in the present study fall within the range of values that have been reported previously in the literature. However, prior to this study, there was no available heme iron database to use in conjunction with dietary data from epidemiologic studies to estimate heme iron intake according to meat type, cooking method and doneness level.
We found that the heme iron content of various meat types varied considerably, even within meat types from the same animal; for example, heme iron values of steak, hamburger and roast beef were different. Differences in iron levels by species, muscle type, age and breed have been documented in the literature [12-14,17,18].
There were no clear effects of cooking method or doneness level on heme iron levels within meat types (Tables 1-3). Previous research has consistently shown that the total iron concentration (μg iron/g meat) increases with cooking (raw versus cooked) and with the level of doneness (cooking temperature) [12-14]; this occurs due to the loss of mass of meat with cooking, while the mass of the relatively non-volatile iron remains constant or may increase as heme iron is degraded at higher temperatures. The concentration of heme iron at any temperature will depend on the loss of meat mass and the degree of degradation of heme iron; this ratio is further complicated by the type and length of cooking, factors that determine the temperature the heme iron experiences. Results from previous studies are mixed, both increases and decreases in the heme iron concentration have been reported [12-15]. Consequently, the lack of a visible trend for heme iron concentration versus doneness in Tables 2-4 is not surprising.
Red meat intake has been associated with a variety of chronic diseases, including diabetes , cardiovascular disease  and cancer  and there are several potential mechanisms involved, including increased exposure to heme iron. Iron overload has many detrimental effects, irrespective of the iron source; therefore, the human body has tight homeostatic control of this trace element. However, the absorption of heme iron is less well regulated than the absorption of non-heme iron [2,3]. In addition to being more readily absorbed than non-heme iron, there are detrimental effects pertaining to heme iron specifically, including cytotoxicity [6,7], and increased endogenous formation of NOCs, which is not affected by nonheme iron intake .
Interest in heme iron intake has been escalating over recent years, leading some investigators to estimate intake of heme iron using proportions of total iron. There are two methods of estimating heme iron: by using 40% of total iron from meat [15,26,27] or by using meatspecific proportions: 69% for beef; 39% for pork, ham, bacon, pork-based luncheon meats, and veal; 26% for chicken and fish; 21% for liver . Although the animal specific proportions are more realistic, our data shows that heme iron values in different types of meat derived
Table 4. Heme iron content (mg/g) of chicken according to meat type, cooking method and doneness level.
from the same animal have varying heme iron contents. Using such estimations has led to inconclusive data for the association between heme iron intake and a variety of cancers. For colon cancer, one study reported an association between heme iron intake and higher risk for proximal colon cancer , another two studies reported suggestive/borderline statistically significant positive associations [16,28], whereas the most recent study was null . In addition, a positive association between heme iron intake and lung cancer risk has been reported , as well as a suggestive, but not statistically significant, positive association for upper digestive tract tumors , but no association for cancer of the breast  or endometrium  in a Canadian cohort of women.
Despite the advantages of being able to estimate heme iron intake using laboratory measurements for a variety of meat types for the first time in epidemiologic studies, there are some limitations to this data. Some of the meat samples were extremely non-homogeneous with regard to fat, gristle, and charred pieces of meat; while the inhouse reference material did not have variations arising from cooking, it did suffer from the same fat and gristle inclusions. Furthermore, all of the meat samples analyzed were bought locally and although many were brand name products, allowing the data to represent nationally available foods, it may not adequately represent meat items from other countries; in addition, the cooking techniques used may not reflect those used in other countries. Finally, each of the measurements on a specific meat type by cooking method and doneness level was made on only one composite sample; therefore, we were unable to estimate the variation within each subcategory.
In conclusion, we report the methodologies used to create a heme iron database that can be used in conjunction with food frequency questionnaires to estimate heme iron intake in relation to disease outcome in epidemiologic studies. We found considerable differences in the heme iron content of various meat types. However, in contrast to the literature, we did not observe an effect of cooking methods or doneness level on the heme-iron content of meat.
AJC and RS are responsible for the design of the study and for writing the manuscript. JMH is responsible for collection and analysis of the data. LMF and AR provided help with the analysis of the data. STM provided significant consultation. Furthermore, we have no financial or personal relationships with the company or organization sponsoring the research at the time the research was done.
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