Efficiency of Hemoglobin Regeneration as a Method of Assessing Iron

1 Efficiency of Hemoglobin Regeneration as a Method of Assessing Iron Bioavailability in Food Products ARTHUR W. MAHONEY and DELOY G. HENDRICKS

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch001

Utah State University, Colleges of Family Life and Agriculture, Department of Nutrition and Food Sciences, Logan, UT 84322

The bioavailability of iron from any source (e.g., iron supplement, food or meal composite) is considered to be that portion of the total iron which is metabolizable. Philosophically, this concept is important because the amount of iron utilized by avian and mammalian species is directly associated with iron need. When assaying iron bioavailability, it is therefore necessary to use an organism whose need will exceed the amount provided. In animal assays of iron bioavailability, iron need is assured by a growth phase and/or creation of iron deficiency through feeding an iron deficient diet and phlebotomy. Because healthy subjects are usually used in human assays of iron bioavailability (Cook et al., 1981; Cook and Monson, 1976; Radhakrishman and Sivaprasad, 1980), i t is inappropriate to compare the data obtained from animal and human assays. In fact i t is questionable i f assays of iron bioavailability yield good information on the quantities of metabolizable iron available when healthy human subjects are used. The Committee on Dietary Allowances, Food and Nutrition Board, National Academy of Sciences (RDA, 1980) has estimated the amount of metabolizable iron (as absorbable iron) from meals consumed by human beings as ranging from 3 to 23 percent depending on the nature of the meal. For adult women of childbearing age, the committee has assumed that 1.5mg iron is lost daily and that 18mg should be consumed to meet this need. They have therefore assumed that approximately 8.3 percent of the dietary iron will be metabolized. For adult men and women over the age of 51 years, they estimate that 1.0mg iron will be lost daily and recommend that 10mg should be consumed to meet this need to offset only approximately 10 percent of the dietary iron being metabolized by these people. It should be noted, however, that what is metabolized from a food under such conditions does not necessarily reflect what is potentially metabolizable. Indeed, the majority of women of childbearing age consume less than the recommended 18mg iron 0097-6156/82/0203-0001$06.0O/0 © 1982 American Chemical Society In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


2

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

and yet are not i r o n d e f i c i e n t (DHEW, 1968-70). Thus, much information i s needed on the m e t a b o l i z a b i l i t y of food i r o n . Two b a s i c methods have been used i n the assay of i r o n b i o a v a i l a b i l i t y (Bing, 1972; Thompson and Raven, 1959). In the absolute method, the change i n t o t a l body i r o n r e l a t i v e to that consumed i s used. T h i s n e c e s s i t a t e s making an estimate of the amount of i r o n present i n the animal body at the i n i t i a t i o n of the experiment and then determining the amount present at the t e r m i n a t i o n . U s u a l l y i n a p p l y i n g t h i s procedure, a r e p r e s e n t a t i v e group of animals i s k i l l e d at the beginning of the experiment to o b t a i n the estimate of t h e i r i n i t i a l body i r o n . Thus, one can o b t a i n an average value f o r body i r o n content r e l a t i v e to weight that can be m u l t i p l i e d with i n i t i a l body weights to estimate i n i t i a l amounts of body i r o n f o r each t e s t animal. Various m o d i f i c a t i o n s of the hemoglobin regeneration procedure have been used(Bing, 1972). In the one described here, the amount of i r o n gained as hemoglobin i s estimated and expressed r e l a t i v e to the amount of i r o n consumed. An e f f i c i e n c y of the conversion of food i r o n i n t o hemoglobin can be computed f o r each t e s t animal knowing i n i t i a l and f i n a l body weights, i n i t i a l and f i n a l hemoglobin c o n c e n t r a t i o n s , the amount of food consumed, and the i r o n content of the food. I t i s c a l c u l a t e d as f o l l o w s : mg Hb Fe

BW x .067 ml b l / g BW X g Hb/100 ml X 3.35mg Fe/g Hb Efficiency = ( ( F i n a l mg Hb Fe - I n i t i a l mg Hb Fe) / mg Fe consumed) X 100

In a p p l y i n g t h i s method, weanling male r a t s are given f r e e access to a low-iron d i e t and b l e d to remove about one ml of blood two times 4 days apart. Three days l a t e r , the animals are again b l e d of about 100 m i c r o l i t e r s blood f o r determination of hemoglobin c o n c e n t r a t i o n and are a l l o t t e d to treatments of ten r a t s each such that mean body weights and hemoglobin concentrations are s i m i l a r . The mean hemoglobin concentrations should be between 4 and 6 gm/dl. They are fed the t e s t d i e t s f o r ten days i n amounts that very few o r t s are obtained. Any s p i l l a g e and o r t s are weighed and recorded to account f o r unconsumed d i e t a r y i r o n . The low-iron d i e t should c o n t a i n l e s s than 10 ppm Fe and the t e s t d i e t s should c o n t a i n approximately 35 ppm. T h i s amount of d i e t a r y i r o n has been shown not to exceed the a b i l i t y of t h i s animal p r e p a r a t i o n to u t i l i z e i r o n , s i n c e the regeneration of hemoblobin i r o n i s l i n e a r at l e a s t to 68 ppm d i e t a r y i r o n (Mahoney and Hendricks, 1976). M i l l e r (1977) reported that i r o n gained as hemoglobin was l i n e a r (r=0.94) through i n t a k e s of 5.5mg i r o n as f e r r o u s s u l f a t e i n 11 days. Her r a t s were made anemic by feeding low-iron d i e t f o r 24 days i n p r e p a r a t i o n f o r the hemoglobin regeneration experiment. The f o l l o w i n g c r i t e r i a f o r a good b i o a v a i l a b i l i t y assay are a p p r o p r i a t e , (a) I t must be dose responsive. For an

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


1.

MAHONEY AND HENDRC IKS

Assay by Hemoglobin Regeneration

assay to be u s e f u l i n a v a r i e t y of s i t u a t i o n s , i t should not be a f f e c t e d by v a r i a t i o n s i n amounts of i r o n consumed. Therefore, the dose-response r e l a t i o n s h i p should be l i n e a r . (b) I t must d i s c r i m i n a t e w i t h good s e n s i t i v i t y among sources of i r o n and among treatments such as cooking o r p r o c e s s i n g . (c) B i o a v a i l a b i l i t y values obtained should be u n a f f e c t e d by f a c t o r s u n r e l a t e d to the food or i r o n source. Thus, the b i o a v a i l a b i l i t y assay should be i n s e n s i t i v e to v a r i a t i o n s i n c a l o r i c d e n s i t y of the d i e t , a p p e t i t e of the animal, and animal maturity, (d) The procedure should y i e l d r e p r o d u c i b l e r e s u l t s f o r the same i r o n source among experiments and l a b o r a t o r i e s . The e f f i c i e n c y of converting d i e t a r y f e r r o u s s u l f a t e i r o n i n t o hemoglobin by anemic r a t s has been c a l c u l a t e d from the data of many experiments and l a b o r a t o r i e s (Table 1 ) . The 'uncorrected' e f f i c i e n c y v a l u e s represent the values obtained f o r the t o t a l amounts of i r o n i n the d i e t s and the ' c o r r e c t e d ' values represent a mathematical e s t i m a t i o n of the hematinic response to only the f e r r o u s s u l f a t e i r o n present i n the d i e t . T h i s estimate was made assuming that the amount of i r o n present i n the low-iron b a s a l d i e t s r e f l e c t s the cumulative i r o n provided by the b a s a l i n g r e d i e n t s of the f e r r o u s s u l f a t e t e s t d i e t s (e. g., c a s e i n , o i l , dextrose, f i b e r , v i t a m i n mixture and m i n e r a l mixture). Thus, knowing the amounts of d i e t consumed by the t e s t animals, one can estimate the c o n t r i b u t i o n of the b a s a l i n g r e d i e n t s to the t o t a l d i e t a r y intake of i r o n of the t e s t animals. T h i s value subtracted from the t o t a l i r o n intake y i e l d s the estimated i r o n intake from f e r r o u s s u l f a t e . S i m i l a r l y , the amount of i r o n gained as hemoglobin by the r a t s f e d the low-iron b a s a l d i e t can be c a l c u l a t e d and subtracted from the t o t a l i r o n gained as hemoglobin by the r a t s f e d the f e r r o u s s u l f a t e t e s t d i e t s , which y i e l d s an estimate of the f e r r o u s s u l f a t e c o n t r i b u t i o n to the i r o n gained as hemoglobin. T h i s v a l u e , r e l a t i v e to the estimated q u a n t i t y of i r o n consumed as f e r r o u s s u l f a t e , was used to compute the ' c o r r e c t e d ' e f f i c i e n c i e s presented i n t a b l e 1. The ' c o r r e c t e d ' values wet^e computed s i m i l a r l y f o r the i r o n sources presented i n t a b l e 2. The v a l i d i t y of t h i s c o r r e c t i o n i s d o u b t f u l when foods are the source of experimental i r o n because the amounts of b a s a l i n g r e d i e n t s are decreased depending on the i r o n content of food t e s t e d , which a f f e c t s the amount of food that must be formulated i n t o the d i e t to provide the d e s i r e d i r o n content. For f e r r o u s s u l f a t e , the average e f f i c i e n c y of c o n v e r t i n g d i e t a r y i r o n i n t o hemoglobin was 52 percent with a c o e f f i c i e n t of v a r i a t i o n of 19 percent (Table 1) . When c o r r e c t e d f o r the b a s a l d i e t a r y i n g r e d i e n t s , the average e f f i c i e n c y was 61 percent, w i t h a c o e f f i c i e n t of v a r i a t i o n of 33 percent. Making the c o r r e c t i o n f o r the b a s a l i n g r e d i e n t s d i d not improve the a n a l y s i s . In two cases, the ' c o r r e c t e d ' e f f i c i e n c y of conversion was g r e a t e r than 100 percent.


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NUTRT IO INAL BO IAVAL IABL IT IY OF R ION Table 1.

E f f i c i e n c y o f Converting I r o n i n FeSO^ i n t o Hemoglobin by Anemic Rats

D i e t a r y Fe 33.0 40.4 31.2 27 27


—

27.8 16.2 20.5 29.2 45.0 18.2 25.6 41.8 68.9 11.8 18.9 23.8 23.6 35.2 48.2

13.8 19.8 31.8 12.2 17.2 22.2 27.2 14 20 32 15 22 32 16.5 26.5 46.5 Mean +_ Sd

Efficiency Uncorrected Corrected** 80 111 50 52 38 42 71 45 69 44 54 56 51 70 47 68 48 60 52 61 46 49 44 33 41 34 46 41 36 39 57 53 62 60 72 71 54 42 60 57 66 66 49 55 53 59 57 62 49 50 52 85 57 76 48 58 53 148 57 89 47 61 33 39 38 45 43 50 42 46 41 58 60 78 53 60 51 71 67 81 57 64 54 74 57 66 43 47 52 + 10 61 + 20 c

Reference Farmer et a l . (1977) A l l r e d (1976) Mahoney e t a l . (1979) Rahotra e t a l . (1973) Anderson e t a l . (1972) Mahoney et a l . (1974) Blumberg & A r n o l d (1947)

Mahoney & Hendricks (1976)

Miller

(1977)

Cardon e t a l . (1980)

Theur e t a l . (1971)

D

Theur e t a l . (1973)°

F r i t z e t a l . (1974)

F r i t z e t a l . (1970)°

Shah e t a l . (1979)

Shah and Belonje (1973a)

Shah and Belonje (1973b)

T-2.67

(P

.02)

Continued on next page.


1.


Table


1-Continued

Note: Uncorrected e f f i c i e n c i e s of 82, 77, 74, 65, 63, 84, 82, and 65 percent were c a l c u l a t e d using data presented by Cowan et a l . (1967). Because there were i n s u f f i c i e n t data published to c a l c u l a t e the c o r r e c t e d e f f i c i e n c i e s , these data were not included i n Table 1. a The e f f i c i e n c y was c o r r e c t e d by estimating the c o n t r i b u t i o n of jlron i n the basal d i e t to i r o n intake and hematinic response. Supplemental data necessary f o r computations s u p p l i e d by authors. Values greater than 100 percent not included i n the mean.


Table 2.

E f f i c i e n c y of Converting Iron From Various Into Hemoglobin By Anemic Rats. D i e t a r y Fe

Source FePO.

Efficiency

18.8

26

4

29.2 54.9 118.0 19.8 31.8 55.8 24.2

18 21 14 22 24 23 21

4 18 12 34 32 27 26

32.6 38.2 49.7 Ground Beef 26.6 Beef Shank 31.0 Beef P l a t e 33.0 Bologna 29.0 Beef 26.0 Turkey 23.0 Turkey 30.4 Enriched Flour 24.4 White Bread 10.7 Whole Wheat Flour 28.0 Rice 28.0 48.0 Dried Egg 21.2

17 23 26 34 63 61 46 49 45 43

18 25 30 42 87 79 62 37 74

24 28

33 49

43 30 43 43

54 31 41 41

FePO,

FePO,

Sources

Reference Blumberg & Arnold (1947)

u F r i t z e t a l . (1974)°

Mahoney & Hendricks (1976)

c

Mahoney e t al.(1974) Farmer e t al.(1977) Farmer e t a l . (1977) , Mahoney e t a l . (1979) Cardon e t a l . (1980) Mahoney e t a l . (1980) Cardon e t a l . (1980) Mahoney e t a l . (1974) M i l l e r (1977)

C

Mahoney e t al.(1974) Shah e t a l . (1979) Mahoney e t al.(1974)

a

The e f f i c i e n c y was c o r r e c t e d by e s t i m a t i n g the c o n t r i b u t i o n of jlron i n the basal d i e t to i r o n intake and hematinic response. Supplemental data necessary f o r computations s u p p l i e d by authors. Due to c a l c u l a t i o n e r r o r s , the o r i g i n a l value was reported as 45 f o r ground beef and 33 f o r whole wheat f l o u r . c


5


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NUTRT IO INAL BO IAVAL IABL IT IY OF R ION In ten cases, the 'corrected* values were l e s s than the uncorrected ones. Because of t h i s i n c o n s i s t e n c y and because c o r r e c t i o n does not reduce v a r i a b i l i t y w i t h i n nor among experiments, attempting to c o r r e c t f o r the i r o n c o n t r i b u t i o n of the b a s a l i n g r e d i e n t s to the hematinic response does not seem to improve t h i s assay of i r o n b i o a v a i l a b i l i t y . D i e t a r y i r o n l e v e l does not seem to a f f e c t the e f f i c i e n c y with which d i e t a r y i r o n i s converted i n t o hemoglobin when f e r r o u s s u l f a t e (Table 1) or when f e r r i c orthophosphate (Table 2) i s the primary source of d i e t a r y i r o n . T h i s i s a l s o true f o r white bread (Table 2); however, the source of the i r o n i n the enriched f l o u r used i n the bread i s unknown. That the e f f i c i e n c y of converting food i r o n i n t o hemoglobin i s not a f f e c t e d by d i e t a r y i r o n c o n c e n t r a t i o n i s important to b i o a v a i l a b i l i t y t e s t i n g because i t i s o f t e n d i f f i c u l t to formulate d i e t s w i t h p r e c i s e amounts of i r o n , e s p e c i a l l y when foods are the sources of i r o n . The e f f e c t s of carbohydrate and f a t on the e f f i c i e n c y with which d i e t a r y i r o n i s converted i n t o hemoglobin have been s t u d i e d . M i l l e r and Landes (1976) used s t a r c h , sucrose or glucose as the carbohydrate source and f e r r o u s s u l f a t e as the i r o n source. The r e s p e c t i v e e f f i c i e n c i e s of converting d i e t a r y i r o n i n t o hemoglobin were 72, 65, and 46 percent. Amine and Hegsted (1971) obtained s i m i l a r carbohydrate e f f e c t s studying i r o n a b s o r p t i o n . Glucose i s the most commonly used source of d i e t a r y carbohydrate i n s e m i p u r i f i e d d i e t s . Pennell et a l . (1976) reported that b e t a - l a c t o s e i n p l a c e of sucrose reduced the r e l a t i v e b i o l o g i c a l value of i r o n as sodium i r o n pyrophosphate when fed to r a t s . However, a l p h a - l a c t o s e or glucose i n p l a c e of the sucrose d i d not a f f e c t the b i o a v a i l a b i l i t y of t h i s i r o n source. S i m i l a r l y , the source of f a t can a f f e c t the b i o a v a i l a b i l i t y of d i e t a r y i r o n ; but, the l e v e l of d i e t a r y f a t has no e f f e c t (Mahoney et a l . , 1980). The c a s e i n c o n c e n t r a t i o n of d i e t s fed r a t s does not a f f e c t i r o n a b s o r p t i o n (Amine and Hegsted, 1971, Carmichael et a l . , 1975); however, e f f e c t of p r o t e i n source was not s t u d i e d by these authors. Thus, the sources of carbohydrate and f a t can markedly a f f e c t the u t i l i z a t i o n of d i e t a r y i r o n and should be considered as important v a r i a b l e s i n b i o a v a i l a b i l i t y experiments. The amount of p r o t e i n , however, does not seem as critical. Among experiments, the v a r i a b i l i t y of the e f f i c i e n c y of converting i r o n from f e r r o u s s u l f a t e i n t o hemoglobin (Table 1) was much g r e a t e r than when f e r r i c orthophosphate (Table 2) was the i r o n source. T h i s v a r i a b i l i t y i s d i s t u r b i n g s i n c e f e r r o u s s u l f a t e i s commonly used as a reference source of i r o n f o r b i o a v a i l a b i l i t y experiments, as w e l l as an i r o n supplement clinically. T y p i c a l l y , t h i s v a r i a b i l i t y i s d e a l t w i t h by expressing the hematinic responses of the unknowns r e l a t i v e to f e r r o u s s u l f a t e (Shah et a l . , 1979; C o c c o d r i l l i et a l . , 1976; Amine et a l . , 1972).



1.



Using the e f f i c i e n c y of c o n v e r t i n g d i e t a r y i r o n i n t o hemoglobin, e f f e c t s of food p r o c e s s i n g procedures on the b i o a v a i l a b i l i t y of i r o n i n meat have been s t u d i e d . Farmer e t a l . (1977) showed that the b i o a v a i l a b i l i t y o f i r o n from mechanically deboned meat was l e s s than that from hand deboned meat; but, more metabolizable i r o n was a v a i l a b l e i n the mechanically deboned product because of i t s g r e a t e r i r o n content. There was no d i f f e r e n c e , however, between the i r o n b i o a v a i l a b i l i t y from mechanically deboned and hand deboned turkey frame meat ( A l l r e d , 1976). The d i f f e r e n c e i n i r o n b i o a v a i l a b i l i t y between the mechanically deboned turkey and the mechanically deboned beef might be a t t r i b u t e d to d i f f e r e n c e s i n abrasiveness o f the meat and bone mixture on the machinery, which would modify the amount and form o f i r o n i n the two products (Farmer e t a l . , 1977). The b i o a v a i l a b i l i t y of meat i r o n i s decreased due t o c u r i n g . T h i s decrease i s dose dependent with n i t r i t e added, u n t i l r e s i d u a l n i t r i t e begins t o accumulate (Mahoney e t a l . , 1979). R e s i d u a l n i t r i t e was a s s o c i a t e d w i t h an apparent i n c r e a s e i n i r o n b i o a v a i l a b i l i t y , which was explained on the b a s i s o f some n i t r i c oxide b i n d i n g t o hemoglobin, rendering a f r a c t i o n o f i t unable t o c a r r y oxygen and thus s t i m u l a t i n g hematopoiesis. Severe atmospheric o x i d a t i o n o f beef r e s u l t s i n depressed i r o n b i o a v a i l a b i l i t y and growth i n r a t s while s i m i l a r o x i d a t i o n of turkey meat d i d not (Cardon et a l . , 1980). Based on the l i m i t e d data a v a i l a b l e , the r e l a t i v e b i o l o g i c a l v a l u e s o f i r o n sources are s i m i l a r whether determined by the s l o p e - r a t i o assay o r by e f f i c i e n c y o f conversion o f d i e t a r y i r o n i n t o hemoglobin (Table 3 ) . The most descrepancies are observed when the r e l a t i v e b i o l o g i c a l value i s estimated by method " c " i n Table 3. Much a d d i t i o n a l research i s r e q u i r e d t o determine the u t i l i t y of the simpler method of e v a l u a t i n g i r o n b i o a v a i l a b i l i t y by e f f i c i e n c y of converting d i e t a r y i r o n i n t o hemoglobin. I t does, however, take l e s s time than the s l o p e - r a t i o n method, apply t o food s t u f f s of r e l a t i v e l y low i r o n c o n c e n t r a t i o n ( I f o n , 1981), provide f o r d i r e c t measurements o f i r o n u t i l i z a t i o n , and apply to human subjects such as blood donors, anemic s u b j e c t s (Norby and S o l w e l l , 1977) and i n f a n t s (Garry, e t a l . , 1981). I t , t h e r e f o r e , has many p o t e n t i a l advantages as means o f evaluating iron b i o a v a i l a b i l i t y .


1

8

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

Table 3.

Comparison of B i o l o g i c a l Values o f D i f f e r e n t Sources R e l a t i v e to Ferrous S u l f a t e


Iron Source FePO.

R e l . B i o l . Value 51 d

FePO, FePO? FePO,

56 44.5 + 4.8°

FePO,

46

FePO FePO? FePO, i n Breakfast C e r e a l FePO, i n Breakfast C e r e a l

14 75 c

b

v

2 3

Enriched F l o u r Enriched F l o u r White Bread Turkey, raw Turkey, raw Whole egg, d r i e d Egg y o l k Beef, raw Beef, cooked

Efficiency of Hemoglobin Regeneration as a Method of Assessing Iron

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