Distribution of Water in Heterogeneous Food and Model Systems

10

D i s t r i b u t i o n of W a t e r i n H e t e r o g e n e o u s

Food

and

M o d e l Systems P. J. LILLFORD, A. H. CLARK, and D. V. JONES

Downloaded by CORNELL UNIV on June 20, 2017 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch010

Unilever Central Resources, Colworth House, Sharnbrook, Bedford, England

In recent years, numerous examples of complex spin-spin relaxation have been reported in tissue. Most prevalent are studies on striated muscles from various sources (1). Belton et a l . (2) proposed three fractions of water in frog muscle. Based on a graphical deconvolution of spin-spin experiments, T2 values of 230 m.sec (15%), 40 m.sec (65%) and 10 m.sec (20%) were

obtained. Derbyshire and Duff report biphasic spin-spin decays in porcine muscle with relative fractions dependent on the degree of rigor (3). Derbyshire and Woodhouse found complex relaxation in fish, porcine, frog and human muscle was confirmed by Hazelwood et a l . (4) and Pintar et a l . (5). The explanation of these NMR phenomena are complicated by the existence of two theories for the behaviour of water in tissue. These are represented by the membrane theory (6) in which intracellular water is assumed to have the properties of ordinary liquid water, but that its solutes may be different from the extracellular solution due to the semi-permeable nature of the intact cell membrane. A later theory due to Ling (7) suggested that intracellular water may be extensively structured due to the high concentration of fixed charges on macromolecules. The latter theory has been challenged by the measurement of diffusion of intracellular water; i.e. NMR methods have shown that the small difference in measured diffusion coefficients between intra and extracellular water can be accounted for by obstruction effects (8). Most authors now agree that the majority of intracellular water is "free" and account for the reduction in the tissue T^ values by assuming that fast exchange occurs between a small "bound" fraction, and the larger "free" fraction. In the absence of structured water and slow exchange, the complex transverse relaxation is more difficult to explain. There is no doubt that the complex behaviour is within the water protons because of the size of resolvably different relaxation processes (2) . Several authors have discussed the origin of the multiexponential transverse decay. Pearson et al (9.) suggested that 0-8412-0559-0/ 80/47-127-177505.00/ 0 © 1980 American Chemical Society

Rowland; Water in Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


178

WATER IN

POLYMERS

the onset of m u l t i e x p o n e n t i a l decay during r i g o r was induced by m o d i f i c a t i o n of exchange r a t e s , or by the e x i s t e n c e of two p h y s i c a l l y separated water phases. The l a t t e r argument was p r e f e r r e d . B e l t o n et a l (2) i n t e r p r e t e d t h e i r three phases i n terms of p r o t e i n bound, i n t r a c e l l u l a r and e x t r a c e l l u l a r water, whereas Hazlewood et a l , i d e n t i f y i n g a s i m i l a r three f r a c t i o n s , argued that the i n t r a c e l l u l a r water was not exchange averaged (k). D i e g e l and P i n t a r suggested that a separate d i s t r i b u t i o n of c o r r e l a t i o n times a s s o c i a t e d w i t h exchange d i f f u s i o n c o n t r i b u t e d to the T^ r e l a x a t i o n ( 5 ) . T h e i r a n a l y s i s showed that t h i s could not be a complete e x p l a n a t i o n of the observed m u l t i p h a s i c behaviour and that some compartmentalisation w i t h i n the t i s s u e was necessary t o e x p l a i n the e f f e c t s . They a l s o argued that since exchange w i t h i n any one compartment was l i k e l y to be f a s t , the assignment of a r e s o l v a b l e phase to the "bound" water f r a c t i o n was not n e c e s s a r i l y v a l i d , s i n c e each compartment contained exchange averaged bound and f r e e water. Some d i f f i c u l t i e s i n i n t e r p r e t a t i o n have been encountered due to the e x i s t e n c e of complex decay i n t r a n s v e r s e r e l a x a t i o n but simple decay i n l o n g i t u d i n a l r e l a x a t i o n . More recent experiments have shown that l o n g i t u d i n a l r e l a x a t i o n processes are a l s o complex when a c c u r a t e measurements are made ( 1 0 ) · A l l of the authors imply t h a t s e p a r a t i o n of water phases probably occurs a t the c e l l u l a r l e v e l . The semi-permeable nature of the c e l l membrane towards ions and s o l u t e s which are capable of r e l a x i n g water protons p r o v i d e s compartments i n which r e l a x a t i o n r a t e s can be s i g n i f i c a n t l y d i f f e r e n t , even when water t r a n s p o r t a c r o s s the membrane i s very r a p i d . Indeed t h i s property of whole t i s s u e has been used i n the development of an NMR method of determining water t r a n s p o r t a c r o s s e r y t h r o c y t e membranes ( 1 1 ) . Complex r e l a x a t i o n i s not however confined t o b i o l o g i c a l t i s s u e . F o l l o w i n g a freeze-thaw c y c l e , non-exponential decay i s observed f o r the water protons i n an agarose g e l (12); and i s a l s o r e a d i l y observed i n meat models made from completely s y n t h e t i c man-made s t r u c t u r e s (13)· In view of the absence of membranes o r any semi-permeable b a r r i e r s i n these wholly f a b r i c a t e d m a t e r i a l s , the general relevance of compartmental i s a t i o n t o the o b s e r v a t i o n of complex r e l a x a t i o n needs to be re-examined. Experimental Procedures and R e s u l t s A

* Methods. Τ measurements were made by the C a r r - P u r c e l l Meiboom-Gill method T l 4 ) . ^ spacings were: agarose 200 jisec; soya f i b r e s 300 j i s e c ; raw and cooked/drained muscle 40 jisec; and cooked muscle 100 j i s e c . 100 scans were accumulated i n each case. T^ measurements were made u s i n g a l 8 0 - TT - 9 0 ° sequence. For each value of , 20 magnetisation values were accumulated. A l l samples were thermostatted a t 1 4 ° . Data were analysed by m u l t i - e x p o n e n t i a l f i t t i n g using a weighted l e a s t squares


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L I L L F O R D ET A L .

Water in Heterogenous

Systems

179


a n a l y s i s , o r by deconvolution a n a l y s i s ( 1 5 ) « B. M a t e r i a l s . Agarose was from Marine C o l l o i d s , Inc. (REX5468), and used without f u r t h e r p u r i f i c a t i o n . A sample o f g e l was prepared by pressure cooking a 4 . 8 wt% d i s p e r s i o n o f agarose i n d i s t i l l e d water f o r 10 min. Approximately 0 . 3 mi o f the s o l u t i o n was p l a c e d i n a 8 mm O.D. NMR tube and g e l l e d by c o o l i n g . A f t e r r e l a x a t i o n measurements on the g e l had been performed, t h e sample was f r o z e n r a p i d l y i n l i q u i d n i t r o g e n , thawed and r e measured. Soya p r o t e i n f i b r e s were s u p p l i e d by C o u r t a u l t L t d . T h e i r analysed composition i s given i n Table I . A s m a l l b l o c k o f f i b r e s ( 0 . 5 g) was cut from the f i b r e tow and p l a c e d i n an NMR tube, w i t h the f i b r e s o r i e n t e d p a r a l l e l t o the tube w a l l . Table I .

A n a l y t i c a l Composition o f C o u r t a u l d Soya F i b r e s

Water

Oil

%

%

Protein χ 6.25)

(Ν

2

67.5

8.5

Ash

NaCl

%

%

2.2

2.1

(Kesp) pH

% 19.2

5-4

Muscle T i s s u e . A sample o f muscle t i s s u e was taken from the L - d o r s i o f a L i n c o l n s h i r e Red H e i f e r post r i g o r . Approximately 0 . 5 g o f t i s s u e was p l a c e d i n an NMR tube w i t h the f i b r e s o r i e n t e d p a r a l l e l t o the tube w a l l , and the t r a n s v e r s e proton r e l a x a t i o n measured immediately. The sample was then immersed i n a water bath a t 85 C f o r 5 min., cooled and remeasured. F i n a l l y , the excess l i q u o r was d r a i n e d from the sample, and the r e l a x a t i o n remeasured. ^" R e s u l t s . Agarose:- In F i g u r e 1, the e f f e c t o f one freeze/thaw c y c l e on the s p i n - s p i n r e l a x a t i o n o f agarose g e l i s shown. For the i n i t i a l homogenous g e l , the r e l a x a t i o n can be s a t i s f a c t o r i l y d e s c r i b e d by a s i n g l e e x p o n e n t i a l process c h a r a c t e r i s e d by a Τ value o f 46 m.sec. A f t e r freeze/thaw, t h e decay was complex and r e q u i r e d a number o f d i s c r e t e e x p o n e n t i a l s f o r i t s adequate d e s c r i p t i o n . The decay was f i t t e d t o 3 and 4 processes w i t h the r e s u l t s g i v e n i n Table I I . Soya p r o t e i n f i b r e s : - Non-exponential r e l a x a t i o n was detected i n both Τ and measurements (Figure 2 ) . The decay was f i t t e d t o 3 and 4 processes w i t h the r e s u l t s given i n Table I I I .



Figure 1.

Transverse relaxation in 4.8 wt% agarose gels. A: homogeneous gel; B: freeze-damaged gel.


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Distribution of Water in Heterogeneous Food and Model Systems

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