Dietary Calcium Exchangeability and Bioavailability - American

1 Dietary Calcium Exchangeability and Bioavailability Evaluation and Potential Uses of an In Vitro Digestion Procedure E. M. WIEN and RUTH SCHWARTZ

Downloaded by 37.9.40.112 on February 16, 2017 | http://pubs.acs.org Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 Use of the in vitro digestion procedure for estimating dietary calcium exchange with an extrinsic isotope should facilitate in vivo absorption studies using the "extrinsic tag" technique. Only a peptic digestion stage is required for exchangeability measurement. Attempts to extend use of the the procedure to measure parameters of bioavailability by including a pancreatic digestion stage were partly successful. In vitro digestion permits study of the chemistry of food calcium under standardized digestion conditions. Investigations discussed include the effects of varying pH, bile salts, enzymes and food substrates on calcium solubility; and post-digestion fractionation of calcium complexes. Before bioavailability per se is estimated in vitro, more direct comparisons between in vivo and in vitro measurements are needed.

The concept of b i o a v a i l a b i l i t y was developed to explain the difference between the t o t a l amount of mineral i n a food and the amount which was used by the i n d i v i d u a l consuming the food. Over the past s i x t y years or more, there have been numerous studies related to dietary calcium requirements and b i o a v a i l a b i l i t y (1,2). As a r e s u l t , much i s known about non-calcium food components which influence the absorption and u t i l i z a t i o n of dietary calcium under experimental conditions. What now i s lacking i s a detailed knowledge of how these factors interact with calcium under normal conditions of ingestion i n meals. We have developed an i n v i t r o digestion procedure, not as a substitute for i n vivo studies, but as a useful adjunct. Our i n i t i a l objective was to develop an i n v i t r o procedure f o r measuring exchangea b i l i t y , the f r a c t i o n of the food mineral which exchanges with an e x t r i n s i c isotope tracer added to the food. This was expected to f a c i l i t a t e the measurement of food mineral absorption i n humans by the e x t r i n s i c tag method. Secondary objectives were to determine i f i n v i t r o mineral s o l u b i l i t y could be used to estimate potential

0097-6156/ 85/ 0275-0001 $06.00/0 © 1985 American Chemical Society

Kies; Nutritional Bioavailability of Calcium ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

2

b i o a v a i l a b i l i t y and to explore ways of using the i n v i t r o procedure to study the interactions of g a s t r o i n t e s t i n a l secretions with dietary mineral and other dietary components which determine mineral s o l u b i l i t y . A number of i n v i t r o digestion procedures have been developed for estimating i r o n b i o a v a i l a b i l i t y (e.g. 3-5) and nonheme iron exchangeability (£). The procedure discussed i n t h i s paper evolved from one of them (4) f o r studying other minerals. Most of the work i n our laboratory has been done with calcium. In t h i s report our o r i g i n a l procedure (7) i s described. Then the published i n vivo - i n v i t r o comparison experiments (8) are b r i e f l y summarized and subsequent investigations on i n v i t r o digestion condi tions and methods for fractionation of calcium from digests are reported.


In V i t r o Digestion Procedure The i n v i t r o digestion procedure i s outlined i n Figure 1. The pepsin, pancreatin and two conjugated b i l e s a l t s mixtures - porcine b i l e extract (BE) and bovine "sodium taurocholate" (TC) - were obtained from Sigma Chemical Co. (St. Louis, MO). The commercial preparations were analyzed f o r mineral content and decontaminated i f necessary before use i n the procedure. Usually BE was used i n experiments discussed i n t h i s paper. In some experiments the digestion was stopped a f t e r peptic digestion. Results of analyses made a f t e r the peptic digestion only w i l l be referred to as " a f t e r the Ρ stage"; r e s u l t s of analyses made a f t e r the complete peptic + pancreatic digestion w i l l be referred to as "after complete digestion" or "after PPa digestion." Exchangeability i s calculated as the r a t i o of s p e c i f i c a c t i v i t i e s (dpm C a / u g Ca) of the mixture and supernatant, when the e x t r i n s i c isotope was added i n ionizable form (Figure 1). Exchangeability values are expressed as decimal f r a c t i o n s . The i n v i t r o estimate of potential a v a i l a b i l i t y was defined, somewhat a r b i t r a r i l y , as calcium s o l u b i l i t y (18,000 χ g supernatant) a f t e r complete digestion. P o t e n t i a l l y available calcium was expressed as a percentage of the t o t a l food calcium (Figure 1). With the exception of a low i n v i t r o calcium s o l u b i l i t y value f o r whole milk, our e a r l i e r data compared reasonably well with calcium b i o a v a i l a b i l i t y information i n the l i t e r a t u r e (7)· 45

Comparison of In Vivo and In V i t r o Measurements The i n v i t r o procedure was tested i n " c r i t i c a l " experiments designed to make direct comparisons of i n vivo and i n v i t r o estimates of exchangeability and potential b i o a v a i l a b i l i t y and to test the use of i n v i t r o exchangeability values i n i n vivo experiments. (8). Three foods which were expected to show d i f f e r e n t l e v e l s of calcium s o l u b i l i t y and exchangeability, c o l l a r d s , soybeans and spinach, were i n t r i n s i c a l l y labeled with C a i n nutrient solution culture. They were used together with Ca as an e x t r i n s i c l a b e l i n both i n v i t r o and i n vivo experiments. The r e s u l t s (8>) showed the expected v a r i a t i o n i n exchangeable Ca among the foods; exchangeability was not complete f o r soy or spinach. However, i n vivo and i n v i t r o exchangeability values were nearly i d e n t i c a l f o r each food. The i n v i t r o exchangeability values after 1+5

4 7



tt5

SN

T

= PPa S o l u b i l i t y =(Ca _ /Ca )(100)

2

l45

Analyses ( Ca, t o t a l Ca)

1 Supernatant (SN-PPa) 1

Centrifuge: 4°C 15 min., 18,000 χ g

Pancreatic Digestion: 37°C, 20 min., shaking

Analyses ( C a , t o t a l Ca) 45

3

N^HC0 to pH 6.8 biie salts to 10 mM pancSeatin to 0.1% (w/v) H 0 to\l5 ml

I PPa Mixture (Τ) 1

+ + + +

F i g u r e 1. I n v i t r o d i g e s t i o n procedure. (Reproduced w i t h p e r m i s s i o n from Ref. 7. C o p y r i g h t 1982 J . N u t r . , American I n s t i t u t e of Nutrition.)

P o t e n t i a l b i o a v a i l a b i l i t y (%)

N

45

pPa


I Supernatant (SN-P) 1

C e n t r i f i ige: 4°C 15 min. 18,000 χ g

Definitions: Special A c t i v i t y (SA) = dpm Ca/ug Ca Exchangeability= SA^/SAg

1+5


< P-Mixture |

f P e p t i c Digest (Ρ) |

2

Peptic D i g e s t i o n : 37°C, 60 min., continuous shaking

+ HC1 to pH 1.5 + peps η to 0.1% (w/v) + H 0 to 9 ml

2

0.5 g dry or freeze-dried food, ground to pass No. 40 sieve (ASMTE-11) + 4.5 g H 0


N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

4

PPa digestion were used to correct i n vivo e x t r i n s i c tag absorption i n order to estimate i n t r i n s i c food calcium absorption. The corrected e x t r i n s i c tag absorption agreed well with the i n t r i n s i c tag absorpt i o n i n rats f o r a l l three foods. When the estimates of b i o a v a i l a b i l i t y were compared (8), i n vivo absorption was higher than i n v i t r o s o l u b i l i t y f o r two of the foods: We had expected absorption to be less than s o l u b i l i t y due to physiolog i c a l factors (1,9). Thus, t h i s surprising result led to the reexamination of i n v i t r o digestion conditions which i s reported i n t h i s paper.


Review of In V i t r o Digestion Conditions Experiments were conducted to determine i f varying the conditions i n the i n v i t r o digestion procedure would affect post-digestion calcium s o l u b i l i t y and i n some cases, exchangeability. This was done with two purposes: to test the use of the i n v i t r o digestion procedure f o r studying factors which might influence calcium b i o a v a i l a b i l i t y and to use the results to modify the standard procedure. Weights and volumes were doubled from the o r i g i n a l procedure (Figure 1) to have more material for analysis. Peptic Digestion Only i n i t i a l pH of the peptic digestion was studied. The pH was set either at 1.5, near the pH optimum f o r pepsin a c t i v i t y (10) or 2.0, to keep the pH closer to the range observed during human gastric digest i o n (11). The r e s u l t s i n Table I show the progressive changes i n calcium "exchangeability" and s o l u b i l i t y from the i n i t i a l s l u r r y through peptic and pancreatic digestion f o r two cow's milk products and four soy products. Varying the peptic pH between 1.5 and 2.0 had l i t t l e effect on exchangeability and s o l u b i l i t y at either the peptic or pancreatic stage. There was l i t t l e relationship between the i n i t i a l slurry and post-digestion values. Exchangeability was determined at the peptic stage, but was incomplete f o r three of the foods. L i t t l e change occurred during pancreatic digestion. S o l u b i l i t y was maximum a f t e r peptic digestion and decreased during pancreatic digest i o n f o r four of the foods. Since the exchangeability did not change during the pancreatic digestion while s o l u b i l i t y decreased, the food calcium and e x t r i n s i c tag must have precipitated from solution at the same rate during pancreatic digestion. Pancreatic Digestion The pancreatic digestion conditions studied included pH, the method of pH control, and b i l e s a l t s mixture and concentration. In addition, experiments were run to determine i f mineral s o l u b i l i t y was affected by enzymatic a c t i v i t y , or only by pH-induced s o l u b i l i t y changes. pH and pH Control. In the o r i g i n a l procedure (Figure 1), the pH was adjusted to pH 6.8 with freshly prepared NaHC0 . The pH generally rose during pancreatic digestion but the magnitude varied with d i f f e r e n t foods and pH adjustment techniques. The data i n Figure 2 are from a number of experiments i n which the pH at the end of pancreatic digestion varied from about 6.2 to 7.2. Although calcium 3



3.46 3.46

4.28 4.28

Defatted Soy flour

Soy Protein Concentrate

G

7.0 7.0

7.1 7.1

6.8 6.8

6. 9 6. 9

6. 7 6.7

I 6.7 6.7

1.5 2.0

1.5 2.0

1.5 2.0

1.5 2.0

1.5 2.0

Po 1.5 2.0

2.2 3.1

1.9 2.8

2.1 2.8

1.8 2.7

1.8 2.4

Ρ 1.8 2.4

PH at !Stage

2

6.3 6.2

6.3 6.3

6.3 6.2

6.4 6.4

6.5 6.5

PPa 6.7 6.6

0.89± .02 0.89± .02

0.46±,.01 0.46± .01

0.51± .01 0.51± .01

0.47± .01 0.47± .01

0.72± .02 0.72± .02

I 0.90± .04 0.90± .04

0.97±. 02 0.98±. 02

0.82±. 02 0.76±. 01

0.81±. 02 0.76±. 01

0.90±. 05 0.86±. 03

1.00±. 03 0.99±. 02

Ρ 1.00±.,02 0.96±. 02

1.02±,.02 1.01±,.00

0.82±,.02 0.76±,.01

0.89± .03 0.79±,.00

0.93± .03 0.89± .02

0.95± .01 0.98± .02

PPa 0.97± .01 1.02± .03

Exchang eab i l i ty,, SA r a t i o 3,4

54.,2±2.6 54.,2±2.6

15.,2±0.3 15.,2±0.3

28.,3±0.7 28.,3±0.7

23.,2±0.4 23..2±0.4

35,,5±1.0 35,,5±1.0

I 64,,7±2.3 64,,7±2.3

94.,3±2.3 96.,5±0.8

80.,8±5.7 68.,3±2.7

71.,5±1.9 65.,1±1.7

81,,1±4.4 83,,9±0.6

98,,4±1.6 96,>4±0.9

Ρ 95,.4±1.8 93,.3±5.0

3

95.• 7±1.9 93,,5±1. 6

52,,2±0. 5 48,,1±1. 0

71,,2±1. 1 66,,6±0. 4

68,,8±2. 2 66,,7±1. 6

48,,0±0. 5 48,,8±0. 8

PPa 93,,7±1. 8 91,,7±1. 7

Solubility, % > 5

In V i t ro Calcium Exchangeab l i l ity and S o l u b i l i t y in Milk and Soy P:roducts as Affected by Stage of Dig estion and I n i t i a 1 Peptic PH

5

3

2

^ g Ca/flask = mg Ca/g dry substrate. I - i n i t i a l slurry; P - i n i t i a l peptic digest, Ρ = at end of peptic digestion and PPa = at end of complete peptic + pancreatic digestion. U n i t s = mean ± S.D. f o r 3 f l a s k s . ^Units of exchangeability: Ratio, s p e c i f i c a c t i v i t y of the mixture: s p e c i f i c a c t i v i t y of the 18,000 χ g supernatant. U n i t s of s o l u b i l i t y (pg Ca i n 18,000 χ g supernatant/yg i n mixture) χ 100.

1.33 1.33

2.23 2.23

Full-fat Soy flour

Soy Protein Isolate

9.72 9.72

1

mg Ca per flask 12.40 12.40

Whole Milk

Skim Milk

Substrate

Table I.



6


lQOh

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

pH

F i g u r e 2. R e l a t i o n s h i p between c a l c i u m s o l u b i l i t y and pH a f t e r complete d i g e s t i o n f o r f o u r soy p r o d u c t s . Key: s o l i d l i n e , f u l l f a t soy f l o u r ; long-dashed l i n e , soy p r o t e i n i s o l a t e ; short-dashed l i n e , soy p r o t e i n c o n c e n t r a t e ; and d o t t e d l i n e , d e f a t t e d soy f l o u r


1.

WIEN A N D SCHWARTZ

An

In Vitro Digestion

1

Procedure


s o l u b i l i t y decreased as pH increased for nearly a l l foods, the rate of change varied among foods, even i n t h i s c l o s e l y - r e l a t e d series ( a l l soy products). Several approaches to c o n t r o l l i n g the pH were t r i e d . These included the use of PIPES, a synthetic buffer with maximum buffering capacity near pH 7 and n e g l i g i b l e binding capacity for minerals (12), and the gradual addition of bicarbonate from a d i a l y s i s tubing "sack" (5.). The method which proved to be most e f f e c t i v e was a procedure i n which CO 2 was bubbled through the digest mixture both during pH adjustment and throughout the pancreatic digestion (13)· With t h i s procedure the pH could be controlled at pH 6.8 ± 0.1. Enzymes. In an e a r l i e r study (14) complete PPa digestion resulted i n calcium s o l u b i l i t y that was at l e a s t 40$ higher than when the peptic digestion step was omitted. I t was not c l e a r whether the enhanced s o l u b i l i t y was due to enzymatic digestion or incubation at acid pH. Therefore, an experiment was run to investigate the r e l a t i v e importance of pH and enzymatic a c t i v i t y . The t e s t substrate was a defatted soy f l o u r . Four minerals, Ca, Mg, Fe and Zn, were measured i n the digests. The r e s u l t s are presented i n Table I I . At the peptic stage, including pepsin increased Fe s o l u b i l i t y , but not Ca s o l u b i l i t y ; Mg Table I I . E f f e c t of Peptic and Pancreatic Enzymes on Mineral S o l u b i l i t y After In V i t r o Digestion Digestion

Final PH 2.1+.0

Ca 82.6±1.7

Peptic

2.6±.0

pH 2, 60 min. + pH 6.8, 30 min. no enzymes

6.7±.l

pH 2, 60 min. no enzymes

% Soluble ±1.2

Fe 2.2±0.7

Zn 101.9±5.3

75.3±0.7

98.0±1.1

17.310.9

93.4±5.4

41.4±3.6

79.0±3.0

19.3±3.5

56.2±4.6

Mg 104

56.2±4.6 Peptic + 44.7±1.2 6.6±.0 48.9±1.2 84.0±1.4 Pancreatic Note: The s u b s t r a t e was a commercial d e f a t t e d soy f l o u r p r o d u c t . Values are mean + S.D. f o r 4 f l a s k s . Each f l a s k c o n t a i n e d 1.0 g substrate. T o t a l m i n e r a l c o n t e n t (\Xglg s u b s t r a t e ) i s Ca, 2778; Mg, 3068; Fe, 87; and Zn, 53. and Zn were completely soluble at pH 2, even without pepsin. A comparison of complete (PPa) digestion with successive non-enzymatic incubations at pH 2 and pH 6.8 indicated that the enzymes greatly increased Fe s o l u b i l i t y and s l i g h t l y increased Ca s o l u b i l i t y . pH and Pancreatic Digestion. The pH range of the i n v i t r o pancreatic digestions (Figure 2) was s i m i l a r to i n vivo conditions, but generally below the pH optima f o r pancreatic enzymes (10,11). To determine i f pH had an i n d i r e c t e f f e c t on calcium s o l u b i l i t y through an e f f e c t on the rate of digestion, protein and carbohydrate digestion and calcium s o l u b i l i t y were measured i n the same PPa digests. The digestions consisted of the standard peptic digestion, followed by pancreatic


8



digestion i n which the pH was set at 6.2, 6.5, 6.8 or 7.1. The substrates were complex foods, muffins, which had been prepared f o r another study (15). When 50% of the bran i n the bran muffin was replaced by freeze-dried spinach or lettuce, the t o t a l calcium i n the muffin was increased by 56% or 65?, respectively. The pH had very l i t t l e effect on carbohydrate or protein digestion, but the calcium s o l u b i l i t y dropped by at least k0% i n a l l three muffin formulations over the 0.9 pH unit increase (Table I I I ) . Calcium s o l u b i l i t y was much lower i n the spinach-containing muffins and dropped more sharply with increasing pH than i n the bran and lettuce-containing muffins. The calcium data i n Table I I I confirm that a small change i n digest pH a f f e c t s the r e l a t i v e calcium s o l u b i l i t i e s from d i f f e r e n t foods. B i l e S a l t s . We used crude conjugated b i l e s a l t s mixtures prepared from b i l e of two species, TC (bovine "sodium taurocholate") and BE (porcine b i l e extract) to determine i f the type of concentration of b i l e s a l t s affected PPa calcium s o l u b i l i t y . Each mixture was used at three concentrations (equal weights), approximately 5, 10 and 15 mM, a l l within the range found i n i n t e s t i n a l contents (11). The substrates were three foods with d i f f e r e n t l i p i d compositions: fullfat soy f l o u r , whole milk and whole egg. The "soluble" calcium i s aqueous calcium and does not include the non-emulsified l i p i d layer v i s i b l e i n some of the lower b i l e s a l t concentrations. The r e s u l t s i n Figure 3 are f o r digestions a t pH 6.8. For soy flour and milk, increasing the concentration of either TC or BE s l i g h t l y decreased soluble calcium and there were no consistent differences between the two b i l e s a l t s mixtures. For egg, both mixtures caused a marked decrease i n calcium s o l u b i l i t y . When the experiment was repeated using a pH maintenance procedure which produced a f i n a l pH of pH 6.5, the pattern of r e s u l t s was s i m i l a r but a l l s o l u b i l i t i e s were 5-15Î higher. Fractionation of Calcium from Digests We have t r i e d two non-destructive approaches to fractionation of the mineral complexes i n i n v i t r o digests: u l t r a f i l t r a t i o n and g e l filtration. Only preliminary data from gel f i l t r a t i o n experiments are currently available. An example of the r e s u l t s of u l t r a f i l t r a t i o n i s presented i n Table IV. The fractionation of the digest mixture, which was prepared from a soy protein i s o l a t e , combines the techniques of centrifugation and u l t r a f i l t r a t i o n . Since the digest contained bicarbonate as the main buffer, C 0 was used to apply pressure to the u l t r a f i l t r a t i o n c e l l , rather that N , to avoid forming p r e c i p i t a t e s . Most of the digest calcium was soluble a f t e r centrifugation at 18,000 χ g. Much of i t was bound to large complexes, as judged from the f i l t r a t i o n of less than h a l f the calcium through the 10,000 MWC0 (molecular weight cut off) membrane. The nominal MWC0 of the membrane i s not a precise guide to the size of complex which i s f i l t e r e d since one-third of the Ca from CaCl2 was retained by the 1000 MWCO membrane, but the d i s t r i b u t i o n of calcium i n the digest was c l e a r l y d i f f e r e n t from that in the completely ionized solution. For a l l u l t r a f i l t r a t i o n s of the in v i t r o digest, the s t a r t i n g material was the 100,000 χ g supernatant in order to avoid confounding the r e s u l t s with sample deterioration. The r e s u l t s were found to be reproducible and not influenced by the 2

2

+ +



551 562 551 554

319

2

108 106 107 108

14

3

Bran muffin peptide maltose

0.62 0.42 0.37 0.37

(Total= 1.36 mg)

542 540 527 517

322

132 130 134 132

24

0.28 0.19 0.08 0.06

(Total2.12 mg)

573 556 545 531

328

116 113 116 112

17

0.92 0.83 0.63 0.47

(Total* 2.25 mg)

Spinach/bran muffin Lettuce/bran muffin peptide maltose Ca peptide Ca Ca maltose mg/g substrate component i n 18,000 χ g supernatant:

3

2

iFreeze-dried foods were ground to pass a 40-mesh sieve. Freeze-dried spinach or lettuce replace 50% of the bran of the bran muffins i n the spinach/bran and lettuce/bran muffins, respectively. Maltose, the endproduct of amylase a c t i v i t y , was measured by the method of Dahlquist (16). Peptide=supernatant protein which was not p r e c i p i t a t e d when t r i c h l o r o a c e t i c acid (TCA) solution was added to 5% TCA (W/V) (17).

pH 6.2 6.5 6.8 7.1

Initial. (no digestion)

Substrate:! Soluble component:

Table I I I . E f f e c t of In V i t r o Pancreatic Digestion pH on Carbohydrate and Protein Digestion and Calcium S o l u b i l i t y


10


\


ο

^^^^

%

—ι

0

5

10

15

0

5

10

15

0

1

5

ι

js

10

F u l l Fat Soy Flour

Whole Milk

Whole Egg

«o

m

m

15

F i g u r e 3. Calcium s o l u b i l i t y : dependence on b i l e s a l t p r e p a r a t i o n and c o n c e n t r a t i o n used i n i n v i t r o d i g e s t i o n (pH 6.8-6.9). Key: Δ, crude bovine sodium t a u r o c h o l a t e ; and • , p o r c i n e b i l e e x t r a c t .


1.

WIEN A N D SCHWARTZ

An in Vitro Digestion

Procedure

Table IV. F r a c t i o n a t i o n o f Calcium i n an I n V i t r o D i g e s t by C e n t r i f u g a t i o n (CE) and U l t r a f i l t r a t i o n (UF) and Comparison w i t h UF R e s u l t s f o r C a C l 2

Fraction

CaCl

2

Solution

3

1

In Vitro Digest *

F i l t e r a b l e Calcium, 7> Digest Mixture CE:18,000 χ g supernatant CE:100,000 χ g supernatant UF:300,000 MWCO f i l t r a t e UF:10,000 MWCO f i l t r a t e UF:5,000 MWCO f i l t r a t e UF:1,000 MWCO f i l t r a t e


1

2

2

2

2

Ca recovery (UF)

1

-

100% 87.4±5.3 67.9±9.1 52.4±6.2 44.9±3.8

95.8% 67.8

30.216.9

98%

93-103%

-

C e n t r i f u g a t i o n at 18,000 χ g, 4°C f o r 15 min. or 100,000 χ g, 4°C for 60 min. U l t r a f i l t r a t i o n was carried out i n an Amicon s t i r r e d c e l l , 65 ml capacity, at 4 C with continuous s t i r r i n g under C0 pressure u n t i l 25 ml f i l t r a t e had been c o l l e c t e d . MWCO = nominal molecular weight cut-off of the membrane. 50 ml of the solution, containing 50 pmoles Ca, was introduced into the UF chamber f o r each determination. Values are f o r duplicate experiments. ^The i n v i t r o digest was prepared by digesting a soy protein i s o l a t e . For each UF determination, 50 ml of the 100,000 g supernatant was introduced into the UF chamber. Values are mean 1 S.D. for four experiments. % f i l t e r a b l e c a l c u l a t i o n : (pg Ca/ml of f i l t r a t e ) / ( y g Ca/ml i n i t i a l solution i n UF chamber) χ 100. The i n v i t r o digest f i l t e r a b l e calcium values were standardized as a % of the o r i g i n a l digest mix ture calcium.

2

2

3

5


12


order of the series of membranes i n a p a r t i c u l a r experiment. Thus, sample deterioration was not a problem when the sample was stored on ice and CO2 saturation maintained. Recovery of the calcium from the combined f i l t r a t e and "retentate" i n the u l t r a f i l t r a t i o n chamber was 93% or better.


Discussion Exchangeability. Measurement of food mineral absorption i n humans by the e x t r i n s i c isotope or e x t r i n s i c tag method i s simpler, more f l e x i b l e and would allow more e f f i c i e n t use of stable isotopes than other methods f o r measuring mineral absorption (7.), i f the r e l a t i o n ship between the absorptions of the e x t r i n s i c tag and i n t r i n s i c food mineral i s known. Cook, et a l . (18) and Hallberg, et a l . (19) o r i g i n a l l y demonstrated the v a l i d i t y of the e x t r i n s i c tag f o r i r o n by feeding i t together with foods labeled i n t r i n s i c a l l y with a second isotope. They r e s t r i c t e d use of the technique to meals i n which there was equal absorption of e x t r i n s i c and i n t r i n s i c iron, that i s , when exchangeability was complete. For other minerals, the requirements for i n vivo v a l i d a t i o n of the e x t r i n s i c tag technique, two "safe" isotopes which can be detected i n the presence of one another and the laborious process of i n t r i n s i c a l l y labeling foods, put severe r e s t r i c t i o n s on the use of the e x t r i n s i c tag technique. Also, a lack of information about the degree of exchange between the e x t r i n s i c tag and test meal i r o n has hampered i n t e r p r e t a t i o n of r e s u l t s from some i r o n absorption studies (20). The i n v i t r o digestion procedure should simplify the use of the e x t r i n s i c tag method. Our r e s u l t s indicate that calcium exchangeabil i t y can be determined i n v i t r o since the i n vivo and i n v i t r o calcium exchangeability values were s i m i l a r (8>). No i n t r i n s i c labeling and only one isotope are required. The same procedure provides the information needed to i n t e r p r e t the r e s u l t s of i n vivo e x t r i n s i c tag studies. Also, i t i s not necessary to demonstrate complete exchangea b i l i t y i n order to use the e x t r i n s i c tag technique f o r measuring calcium absorption. I f exchangeability i s known, even i f i t i s not complete i t can be used to calculate i n t r i n s i c food mineral absorption from the e x t r i n s i c tag absorption (8). Hallberg and Bjorn-Rasmussen (6) reached a s i m i l a r conclusion i n t h e i r studies on absorption of "contamination" i r o n . Measurement of calcium exchangeability i s further s i m p l i f i e d i n that only the peptic digestion step of the i n v i t r o procedure i s required since exchangeability does not change a f t e r that (Table I ) . While our data indicate that i n v i t r o digestion provides a simple means to solve some d i f f i c u l t problems of using the e x t r i n s i c tag method f o r measuring calcium absorption, our conclusions are based on a limited number of foods. They would be strengthened i f a wider range of foods i s tested i n direct i n vivo - i n v i t r o comparison studies. The test foods should include foods i n which exchangeability might not be completed during peptic digestion such as foods with " i n d i g e s t i b l e " residues that may be altered and release calcium which may be absorbed i n the lower i n t e s t i n e . Bioavailability. In p r i n c i p l e , the i n v i t r o procedure provides a r e l a t i v e l y fast and inexpensive means to study calcium b i o a v a i l a b i l i t y as a c h a r a c t e r i s t i c of foods. A knowledge of the chemistry of


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the foods themselves i s not s u f f i c i e n t since changes occur during the process of digestion and the t o t a l chemical environment influences the r e l a t i v e binding constants f o r the various complexes which may be formed (21,22). At the same time, the host c h a r a c t e r i s t i c s which r e s t r i c t i n vivo absorption and introduce v a r i a b i l i t y not related to food c h a r a c t e r i s t i c s (1,2,9) are eliminated. Choice of Potential B i o a v a i l a b i l i t y C r i t e r i o n . I t i s usually assumed that calcium must be soluble and probably ionized i n order to be available f o r absorption (£). For the i n v i t r o procedure, as a f i r s t approximation we chose calcium s o l u b i l i t y a f t e r centrifugation at 18,000 χ g as the measure of potential b i o a v a i l a b i l i t y (Figure 1). We assumed that t h i s would probably overestimate the available calcium and l a t e r work based on f r a c t i o n a t i o n might define the bioavailable calcium more p r e c i s e l y . The data i n Table IV i l l u s t r a t e how the choice of c r i t e r i o n f o r " s o l u b i l i t y " could a f f e c t the i n v i t r o estimate of potential a v a i l a b i l i t y , even i f i n v i t r o conditions c l o s e l y resembled i n vivo conditions. Since our i n v i t r o c r i t e r i o n unexpectedly underestimated calcium b i o a v a i l a b i l i t y f o r two of the three foods i n the direct i n vivo - i n v i t r o comparison (8), i t was necessary to determine the i n v i t r o digestion conditions which might be l i m i t i n g s o l u b i l i t y before addressing the choice of appropriate criterion. Digestion Conditions. Peptic conditions were not emphasized since calcium s o l u b i l i t y i s high at the peptic stage (Table I) and chyme release to the duodenum i s more dependent on p a r t i c l e size than completeness of digestion (23,24). Analyses of the pancreatic digestion conditions indicate that the pH of the pancreatic digest was more important for determining calcium s o l u b i l i t y than enzymatic a c t i v i t y (Figure 2, Tables I I and III) or b i l e s a l t s (Figure 3) for most foods tested. In e a r l i e r studies (7,8) the pH increased 0.2-0.7 pH u n i t s during the pancreatic digestion, presumably due to a combination of bicarbonate decomposition, digestion endproduct release and the variable buffering capacity of the foods. Even though these pH's are i n the range found i n the small i n t e s t i n e (11), the observed pH d r i f t occured i n a range which i s c r i t i c a l for calcium s o l u b i l i t y (£). The f i n a l pH was apparently too high to result i n the net effect seen i n vivo (8). "Standard" Digestion Conditions. As a result of the analyses of digestion conditions we have modified our i n v i t r o digestion to s t a r t the peptic digestion at pH 2.0 instead of 1.5, and to control the pH at 6.8 i n the pancreatic stage by continuous aeration with CO . We also substituted b i l e extract f o r taurocholate since, although calcium s o l u b i l i t y was similar, other minerals were more soluble i n the b i l e extract-containing digests (13)· The term "standard" i s not meant to denote a digestion procedure which should be routinely used to determine p o t e n t i a l l y available calcium. Since the number of foods tested so f a r i s l i m i t e d , i t w i l l be more useful to think of the "standard" procedure as a set of conditions to be used to see how well we understand food chemistry and calcium s o l u b i l i t y i n the g a s t r o i n t e s t i n a l environment. I t should be used f o r measuring the r e l a t i v e s o l u b i l i t y of calcium from foods and meals, but mostly i n the context of comparisons with i n vivo r e s u l t s to define factors which require further study.



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Lipid-containing Foods. The decreased calcium s o l u b i l i t y i n the presence of b i l e s a l t s (Figure 3) suggests factors which require further study. B i l e s a l t s have been shown to enhance calcium absorp t i o n from sparingly soluble s a l t s (25) but also enhance lipase a c t i v i t y (26). Since no absorption occurs i n the i n v i t r o system, the l i b e r a t e d f a t t y acids could form insoluble calcium soaps (27). In contrast, i n vivo calcium absorption i s not i n h i b i t e d by even large amounts of f a t i n the d i e t unless there i s a pre-existing malabsorp t i o n problem (1,2,9). Presumably, l i p i d hydrolysis products are absorbed from the i n t e s t i n e f a s t enough to prevent insoluble calcium soap formation. The complex l i p i d s of egg yolk (28), and t h e i r possible e f f e c t s on l i p a s e a c t i v i t y (29) may explain the marked decrease i n egg calcium s o l u b i l i t y with increasing b i l e s a l t s concen t r a t i o n . More information i s needed to determine i f t h i s i s a problem i n vivo. In any case, these questions should not prevent the use of the i n v i t r o procedure f o r measuring exchangeability i n l i p i d containing foods, since exchangeability i s determined at the peptic stage, as shown f o r whole milk and f u l l - f a t soy f l o u r i n Table I. Fractionation of Digest Calcium. The i n v i t r o digestion procedure provides a means of producing the s t a r t i n g material for a more detailed study of the calcium complexes i n i n t e s t i n a l digests. This may be desirable i n a number of s i t u a t i o n s : 1 . To determine how the degree of exchange between an e x t r i n s i c isotope and the i n t r i n s i c calcium i n the food or meal i s affected by the method of incorporating the isotope, the calcium source i t s e l f or foods fed with i t . 2. To determine how controlled manipulation of digestion conditions influences the d i s t r i b u t i o n of calcium among possible ligands from a food or meal. 3 . To describe the calcium complexes formed during a standardized digestion for a number of foods and food mixtures, f o r comparisons, or to test hypotheses r e l a t i n g food components to i n t e s t i n a l calcium complexes. Fractionation Methods. U l t r a f i l t r a t i o n and gel f i l t r a t i o n are non destructive methods which, based on l i m i t e d experience, can be used for fractionation of mineral complexes from digests. In e a r l i e r studies mineral absorption on the g e l material was a problem. Lonnerdal ( 3 0 ) introduced a method of treating dextran gels with sodium borohydride i n order to eliminate the mineral-binding s i t e s on the g e l . In preliminary studies we have recovered more than 90% of Ca, Mg, Fe, Zn and Ρ i n samples applied to a borohydride-treated gel column (Sephadex G-50, Pharmacia Fine Chemicals, Piscataway, NJ). Recovery of Ca (Table IV) and Mg, Fe and Zn from u l t r a f i l t r a t i o n was also good. In our experience with u l t r a f i l t r a t i o n , use of C 0 pressure to force the f i l t e r a b l e material through the membrane i n the u l t r a f i l t r a t i o n procedure introduced a possible source of error. The high pressure caused more C 0 to be dissolved i n the digest supernatant and the pH i n the chamber decreased to about 6.2. This may have caused a s h i f t of the mineral among ligands ( 3 1 ) . I t should be possible to formulate a mixture of C02 and N2 to maintain the pH i n the chamber, but we have not pursued t h i s . 2

2


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Gel f i l t r a t i o n may be best used to analyze fractions already separated from a digest supernatant by u l t r a f i l t r a t i o n , as used i n a recent study by Sandstrom, et a l . (32). A more precise separation of complexes can be obtained with gel f i l t r a t i o n , but the size of sample which can be applied i s l i m i t e d . Thus, i n many situations, the sample must be concentrated before being applied to the gel column. E i t h e r p r e - p u r i f i c a t i o n or sample concentration could introduce possible s h i f t s i n mineral binding which should be understood f o r proper interpretation of the r e s u l t s (33).


Summary Use of an i n v i t r o simulated g a s t r o i n t e s t i n a l digestion procedure i n calcium b i o a v a i l a b i l i t y research has been discussed. Two d i s t i n c t types of uses were described: (1) measurement of exchangeability to f a c i l i t a t e dietary calcium absorption studies, and (2) study of the fate of food calcium i n the g a s t r o i n t e s t i n a l environment with regard to i t s potential a v a i l a b i l i t y for absorption. Ideas have been incorporated from many sources and only limited testing has been possible so f a r . We have t r i e d to indicate the advantages of such a procedure as well as where more testing of the v a l i d i t y of the ideas for calcium b i o a v a i l a b i l i t y research i s required.

Acknowledgments The research was supported i n part by grants from NIH Grant 18569 and USDA Cooperative Agreement 58-320A4-9-91.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Allen, L.H. Am. J. Clin. Nutr. 1982, 35, 783-808. Irwin, M.I.; Kienholz, E.W. J. Nutr. 1973, 103, 1019-95. Jacobs, Α.; Greenman, D.A. Brit. Med. J. 1969, 1, 673-6. Narasinga Rao, B.S.; Prabhavathi, T. Am. J. Clin. Nutr. 1978, 31, 169-75. Miller, D.D.; Schricker, B.R.; Rasmussen, R.R.; Van Campen, D. Am. J. Clin. Nutr. 1981, 34, 2248-56. Hallberg, L.; Bjorn-Rasmussen, E. Am. J. Clin. Nutr. 1981, 34, 2808-15. Schwartz, R.; Belko, A.Z.; Wien, E.M. J. Nutr. 1982, 112, 497504. Wien, E.M.; Schwartz, R. J. Nutr. 1983, 113, 388-93. Wilkinson, R. In "Calcium, Phosphate and Magnesium Metabolism"; Nordin, B.E.C., Ed.; Churchill-Livingston: Edinburgh, 1976; pp. 36-112. Harper, H.A. "Review of Physiological Chemistry"; Lange: Los Altos, 1975, 15th ed.; p. 230. Fordtran, J.S.; Locklear, T.W. Am. J. Digest. Dis. 1966, 11, 503-21. Good, N.E.; Winget, G.D.; Winter, W.; Connolly, T.N.; Izawa, S.; Singh, R.M.M. Biochemistry 1966, 5, 467-77.



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13. Schwartz, R. 1984, In preparation. 14. Belko, A.Z. M.S. Thesis, Cornell University, Ithaca, 1980. 15. Schwartz, R.; Spencer, H.; Welsh, J.E. Am. J. Clin. Nutr. 1984, 39, 571-6. 16. Dahlquist. A. Scand. J. Clin. Lab. Invest. 1962, 14, 145-51. 17. Layne, E. In "Methods in Enzymology"; Colowick, S. P.; Kaplan, N.O. Eds.; Academic: New York, 1956, Vol. III., pp. 448-50. 18. Cook, J.D.; Layrisse, M.; Martinez-Torres, C.; Walker, R.; Monsen, E.; Finch, C.A. J. Clin. Invest. 1972, 51, 805-15. 19. Hallberg, L.; Bjorn-Rasmussen, E. Scand. J. Haematol. 1972, 9, 193-7. 20. Consaul, H.R.; Lee, K. J. Agr. Food Chem. 1983, 31, 684-9. 21. Leigh, M.J.; Miller, D.D. Am. J. Clin. Nutr. 1983, 38, 202-13. 22. Schubert, J. In "Iron Metabolism"; Gross, F., Ed.; SpringerVerlag: Berlin, 1964; pp. 466-94. 23. Davenport, H.W. "Physiology of the Digestive Tract"; Yearbook Medical Publ.: Chicago, 1982, 5th ed. 24. Arnold, J.G.; Dubois, A. Digest. Dis. Sci. 1983, 28, 737-41. 25. Webling, D. D'A.; Holdworth, E.S. Biochem. J. 1966, 100, 65260. 26. Rathelot, J.; Julien, R.; Canioni, P.; Coeroli, C.; Sarda, L. Biochemie 1975, 57, 1117-22. 27. Patton, J.S.; Carey, M.C. Science 1979, 204, 145-8. 28. Parkinson, T.L. J. Sci. Fd. Agric. 1966, 17, 101-11. 29. Patton, J.S.; Carey, M.C. Am. J. Physiol. 1981, 241, G328-36. 30. Lonnerdal, B. In "Trace Element Analytical Chemistry in Medicine and Biology"; Bratter, P.; Schramel, P. Eds.; WalterDeGruyter: Berlin, 1980; pp. 439-46. 31. Danielson, B.G.; Pallin, E.; Sohtell, M. Uppsala J. Med. Sci. 1982, 87, 43-53. 32. Sandstrom, B.; Keen, C.L.; Lonnderdal, B. Am. J. Clin. Nutr. 1983, 38, 420-8. 33. Chaberek, S.; Martell, A.E. "Organic Sequestering Agents"; John Wiley: New York, 1959; p. 101. RECEIVED October 15, 1984


Dietary Calcium Exchangeability and Bioavailability - American

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