J .

H y g . ,
Camb. ( 1 9 7 1 ) , 6 9 , 1
P r i n t e d i n G r e at B r i t ai n
The c o mp o s i t i o n o f t e a i n f u s i o n s e xami n e d i n r e l at i o n t o t he
as s o c i at i o n be t w e e n mo r t al i t y an d w at e r har d n e s s
B y W. ANDERSON, J . G . H OLLINS* AND P AMELA S. B OND
De p ar t me n t o f P hy s i c s , In s t i t u t e o f Can c e r Re s e ar c h, Cl i f t o n Av e n u e ,
B e l mo n t , Su t t o n , Su r r e y
( Re c e i v e d 2 7 J u l y 1 9 7 0 )
SUMMARY
Re c e n t e p i d e mi o l o g i c al s t u d i e s hav e s ho w n t hat d e at h- r at e s f r o m c e r t ai n
c hr o n i c d i s e as e s ar e hi g he r i n ar e as w i t h s o f t t han i n ar e as w i t h har d d r i n k i n g -
w at e r . In t he s t r i k i n g n e g at i v e c o r r e l at i o n f o u n d i n t he c o u n t y bo r o u g hs o f
En g l an d an d Wal e s be t w e e n c ar d i o v as c u l ar mo r t al i t y an d w at e r har d n e s s t he
i mp o r t an t u n d e r l y i n g f ac t o r i s ap p ar e n t l y t he w at e r c al c i u m. In t e r e s t i s t he r e f o r e
f o c u s e d o n t he d i e t ar y s i g n i f i c an c e o f c al c i u m p r e s e n t i n d r i n k i n g - w at e r . In
r e l at i o n t o t hat i n t e r e s t , t he p r e s e n t r e p o r t g i v e s a q u an t i t at i v e ac c o u n t o f t he
c o mp o s i t i o n o f t e a i n f u s i o n s p r e p ar e d w i t h w at e r s c o n t ai n i n g d i f f e r e n t amo u n t s o f
c al c i u m. It i s s ho w n t hat a s u bs t an t i al p ar t o f w at e r c al c i u m i s t ak e n u p by t he t e a
l e af d u r i n g t he p r e p ar at i o n o f i n f u s i o n s . The an al y s i s o f t he i n f u s i o n s c o v e r s a w i d e
r an g e o f i n d i v i d u al c o mp o n e n t s , i n c l u d i n g t r ac e me t al s an d p o l y p he n o l i c s u b-
s t an c e s . It ap p e ar s t hat t he p r i n c i p al c han g e c au s e d i n i n f u s i o n c o mp o s i t i o n by t he
p r e s e n c e o f c al c i u m i n t he w at e r i s a s u bs t an t i al r e d u c t i o n i n t he r e l at i v e l y hi g h
o xal at e c o n t e n t . The q u e s t i o n i s r ai s e d w he t he r t he r e may be s o me c o n n e xi o n
be t w e e n t he ' w at e r f ac t o r ' i n c ar d i o v as c u l ar d i s e as e an d t he abs o r p t i o n o f o xal at e s
f r o m f o o d s .
INTRODUCTION
At t e n t i o n has be e n d i r e c t e d i n r e p o r t s f r o m s e v e r al c o u n t r i e s t o s t r i k i n g c o r r e l a-
t i o n s w hi c h s ho w t hat i n ar e as s u p p l i e d w i t h s o f t d r i n k i n g - w at e r t he d e at h- r at e s
f r o m c ar d i o v as c u l ar d i s e as e ar e g e n e r al l y hi g he r t han t ho s e i n ar e as s u p p l i e d w i t h
har d w at e r ( K o bay as hi , 1 9 5 7 ; Sc hr o e d e r , 1 9 6 0 ; Mo r r i s , Cr aw f o r d & H e ad y , 1 9 6 1 ,
1 9 6 2 ; B i d r c k , B o s t r o m & Wi d s t r 6 m, 1 9 6 5 ) . Ev i d e n c e has al s o be e n r e p o r t e d
( Tu r n e r , 1 9 6 2 ) o f s i mi l ar n e g at i v e c o r r e l at i o n s be t w e e n w at e r har d n e s s an d mo r t al i t y
f r o m bo t h g as t r i c c an c e r an d p r i mar y bo n e t u mo u r s i n En g l an d an d Wal e s ,
al t ho u g h w i t h f u l l e r d at a t he as s o c i at i o n i n t he c as e o f g as t r i c c an c e r w as l at e r
f o u n d t o hav e o n l y l o w s t at i s t i c al s i g n i f i c an c e ( An d e r s o n , 1 9 6 4 ) . Mo r e r e c e n t l y
Mo r r i s an d hi s c o l l e ag u e s ( Cr aw f o r d , G ar d n e r & Mo r r i s , 1 9 6 8 ) i n a t ho r o u g h e p i -
d e mi o l o g i c al s t u d y c o v e r i n g t he p e r i o d 1 9 5 8 - 6 4 hav e c o n f i r me d t hat f o r t he c o u n t y
bo r o u g hs o f En g l an d an d Wal e s t he as s o c i at i o n be t w e e n w at e r har d n e s s an d
c ar d i o v as c u l ar mo r t al i t y i s p ar t i c u l ar l y mar k e d an d ap p ar e n t l y u n r e l at e d t o
*
P r e s e n t ad d r e s s : Di v i s i o n o f B i o l o g y , Nat i o n al Re s e ar c h Co u n c i l o f Can ad a, Ot t aw a,
Can ad a.
I IIYG 6 9
W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
s o c i al o r o t he r e n v i r o n me n t al f ac t o r s . The s e w o r k e r s c o n c l u d e t hat t he r e i s an
u r g e n t n e e d f o r f u r t he r d e t ai l e d i n v e s t i g at i o n o f t hi s p r o bl e m.
In at t e mp t i n g t o r e l at e t he s e as s o c i at i o n s be t w e e n mo r t al i t y an d w at e r har d n e s s
t o c he mi c al e n t i t i e s p r e s e n t i n d r i n k i n g - w at e r , s e v e r al au t ho r s hav e p o i n t e d t o
t r ac e me t al s i n d r i n k i n g - w at e r as p o s s i bl y r e l e v an t . It ap p e ar s , ho w e v e r , t hat f o r
t he as s o c i at i o n s f o u n d i n En g l an d an d Wal e s t he i mp o r t an t u n d e r l y i n g f ac t o r i s
t he w at e r c al c i u m. In p ar t i c u l ar , i t has be e n s ho w n t hat c ar d i o v as c u l ar mo r t al i t y i s
e v e n mo r e hi g hl y as s o c i at e d w i t h w at e r c al c i u m ( t he l o w e r t he c al c i u m c o n t e n t t he
hi g he r t he mo r t al i t y ) t han w i t h w at e r har d n e s s , an d t he r e i s n o e v i d e n c e o f s i g n i f i -
c an t i n t e r r e l at i o n w i t h an y o t he r w at e r c o mp o n e n t s t u d i e d ( H o l l i n s , 1 9 6 5 ;
Cr aw f o r d e t al . 1 9 6 8 ) . The i mp o r t an t q u e s t i o n r ai s e d i s ho w mi g ht t he c al c i u m
p r e s e n t i n d r i n k i n g - w at e r p e r f o r m a p r o t e c t i v e r o l e .
In har d - w at e r ar e as o f En g l an d an d Wal e s t he c al c i u m c o n t r i bu t i o n f r o m d r i n k i n g -
w at e r s t i l l f o r ms o n l y abo u t a t e n t h o f t he av e r ag e d i e t ar y c al c i u m i n t ak e ( H o l l i n g s -
w o r t h, 1 9 5 6 ) . Cal c i u m d e f i c i e n c y i s n o t o r d i n ar i l y e n c o u n t e r e d i n man ( B r o n n e r ,
1 9 6 4 ) s o t hat i t s e e ms u n l i k e l y t hat t he s mal l c o n t r i bu t i o n o f c al c i u m f r o m d r i n k i n g -
w at e r f u n c t i o n s as an e s s e n t i al s u p p l e me n t o f d i e t ar y c al c i u m f o r an y s u bs t an t i al
p ar t o f t he p o p u l at i o n i n t he s e c o u n t r i e s . At t e n t i o n i s t he r e f o r e t u r n e d t o t he
p o s s i bl e w ay s i n w hi c h c al c i u m f r o m d r i n k i n g - w at e r may e i t he r mo d i f y t he c o m-
p o s i t i o n o r i n f l u e n c e t he abs o r p t i o n o f c e r t ai n d i e t ar y c o mp o n e n t s . In t hi s c o n -
n e xi o n , s i n c e a maj o r p ar t o f t he c u l i n ar y u s e o f w at e r i s as s o c i at e d w i t h t he c o o k i n g
o f v e g e t abl e mat e r i al s an d p ar t i c u l ar l y w i t h t he p r e p ar at i o n o f be v e r ag e s s u c h as
t e a an d c o f f e e , i n f o r mat i o n o n t he i n t e r ac t i o n s o f w at e r c al c i u m an d t he c o mp o n e n t s
o f t he s e mat e r i al s i s o f p r i me i n t e r e s t . F u r t he r i n t e r e s t i s d i r e c t e d t o t he c o mp o s i -
t i o n o f t e a i n f u s i o n s by t he d e mo n s t r at i o n , i n s t u d i e s w i t h l abo r at o r y an i mal s , t hat
s e v e r al v e g e t abl e t an n i n e xt r ac t s p o s s e s s c ar c i n o g e n i c p r o p e r t i e s ( K i r by , 1 9 6 0 ;
K o r p as s y , 1 9 6 1 ) an d al s o t hat t e a e xt r ac t s p o s s e s s c o - c ar c i n o g e n i c p r o p e r t i e s
( K ai s e r , 1 9 6 7 ) .
As a c o n t r i bu t i o n t o t hi s f i e l d , w e hav e e xami n e d q u an t i t at i v e l y t he i n t e r ac t i o n s
w hi c h o c c u r be t w e e n c al c i u m i n w at e r an d t he c o mp o n e n t s o f t he t e a l e af d u r i n g
t he p r e p ar at i o n o f i n f u s i o n s , w i t h at t e n t i o n g i v e n bo t h t o t he mo v e me n t s o f
c al c i u m be t w e e n t he e xt r ac t i n g w at e r an d t he l e af , an d t o t he i n f l u e n c e o f w at e r
c al c i u m o n t he c o mp o s i t i o n o f t he i n f u s i o n s . In t he p r e s e n t c o mmu n i c at i o n w e
s u mmar i z e t he r e s u l t s o f t he s e s t u d i e s .
MATERIALS AND METH ODS
P r e p ar at i o n o f t e a i n f u s i o n s
The s i x s amp l e s o f bl ac k t e a u s e d i n t he p r e s e n t s t u d i e s w e r e o bt ai n e d i n 1 k g .
q u an t i t i e s f r o m a t e a br o k e r , an d c o mp r i s e d t w o bl e n d s o f t e as f r o m Ce y l o n ( A, B ) ,
t w o bl e n d s o f t e as f r o m In d i a ( C, D) , an As s am t e a ( E) , an d a Chi n a t e a ( F ) . An
o o l o n g t e a f r o m F o r mo s a ( G ) an d a g r e e n ( ' g u n p o w d e r ' ) t e a f r o m Chi n a ( H ) w e r e
al s o i n c l u d e d i n s o me o f t he w o r k . The s amp l e s , w hi c h had mo i s t u r e c o n t e n t s
r an g i n g f r o m 3 - 7 t o 5 ' 2
% ,
w e r e s t o r e d i n ai r - t i g ht g l as s j ar s .
The r e l at i v e amo u n t s o f l e af an d w at e r u s e d i n o r d i n ar y d o me s t i c p r ac t i c e by
1 0 i n d i v i d u al s f o r p r e p ar i n g t e a i n f u s i o n s w e r e me as u r e d an d f o u n d t o v ar y be t w e e n
2
Te a i n f u s i o n s an d w at e r har d n e s s
8 an d 2 1 g . d r y l e af / I. w at e r . The me an v al u e o f 1 5 g . l e af p e r l i t r e o f w at e r w as
s e l e c t e d f o r u s e t hr o u g ho u t t he p r e s e n t s t u d i e s . * A s t an d ar d i n f u s i o n p e r i o d o f
6 mi n . w as u s e d s i n c e , w i t h t he e xc e p t i o n o f t he p e c t i n i c ac i d s w hi c h d i s s o l v e d
s l o w l y , t he e xt r ac t i o n o f al l t he c o mp o n e n t s s t u d i e d w as e s s e n t i al l y c o mp l e t e w i t hi n
t hi s p e r i o d . The i n f u s i o n s w e r e p r e p ar e d i n t ar e d g l as s be ak e r s by mi xi n g t he
l e af w i t h bo i l i n g w at e r an d mai n t ai n i n g t he mi xt u r e at 9 5 0 C f o r 6 mi n . Smal l
ad d i t i o n s o f d i s t i l l e d w at e r w e r e mad e as r e q u i r e d t o o f f s e t e v ap o r at i o n l o s s e s .
Co n t r o l t e s t s s ho w e d t hat n e i t he r t he amo u n t o f c al c i u m t ak e n u p by t he l e af , n o r
t he e xt r ac t i o n o f t he p r i n c i p al c o mp o n e n t s f r o m t he l e af , w as me as u r abl y af f e c t e d
by g e n t l e s t i r r i n g , an d , f o r c o n v e n i e n c e i n s amp l i n g f o r t he s t u d y o f u p t ak e an d
e xt r ac t i o n r at e s , t he i n f u s i o n s w e r e g e n e r al l y s t i r r e d g e n t l y d u r i n g t he e xt r ac t i o n
p e r i o d . F o r t he o xal at e s t u d i e s , t he p r o c e d u r e u s e d c o n f o r me d t o o r d i n ar y d o me s t i c
p r ac t i c e w i t h br i e f s t i r r i n g o n l y at t he s t ar t an d f i n i s h o f e xt r ac t i o n . Af t e r e xt r ac -
t i o n , t he i n f u s i o n s w e r e s e p ar at e d f r o m t he l e av e s by p o u r i n g t hr o u g h e i t he r a n y l o n
t e a s t r ai n e r o r a p ad o f g l as s w o o l .
An al y t i c al p r o c e d u r e s
A. R. g r ad e r e ag e n t s w e r e u s e d t hr o u g ho u t .
In o r g an i c c o mp o n e n t s
Ext e n s i v e u s e w as mad e o f e mi s s i o n f l ame s p e c t r o p ho t o me t r y f o r e l e me n t d e t e r -
mi n at i o n , e mp l o y i n g a s p e c i al l y c o n s t r u c t e d f l ame s p e c t r o p ho t o me t e r , bas e d o n
a Z e i s s t o t al c o n s u mp t i o n bu r n e r , a ' Uv i s p e k ' mo n o c hr o mat o r , an d an E. M. I. 1 3 -
s t ag e p ho t o mu l t i p l i e r ( H o l l i n s , 1 9 6 5 ) . The me t ho d s u s e d w e r e c ap abl e o f d e t e c t i n g
mo r e t han f o r t y me t al s at a l e v e l o f 0 5 mg . me t al / 1 5 g . t e a l e af , t he s e me t al s
i n c l u d i n g t he maj o r i t y o f t he G r o u p I an d G r o u p II me t al s , t he r ar e e ar t hs , an d t he
t r an s i t i o n e l e me n t s o f p e r i o d IV o f t he p e r i o d i c t abl e . The c al i br at i o n c u r v e s o f t he
e l e me n t s an al y s e d w e r e l i n e ar o v e r t he r an g e o f c o n c e n t r at i o n s u s e d an d t he s t an -
d ar d ad d i t i o n t e c hn i q u e ( Cho w & Tho mp s o n , 1 9 5 5 ) w as u s e d t o s t an d ar d i z e
me as u r e me n t s o n al l s o l u t i o n s . Me as u r e me n t s w e r e mad e e i t he r d i r e c t l y o n s u i t abl e
d i l u t i o n s o f t he i n f u s i o n s o r o n s amp l e s o f i n f u s i o n o r l e af w hi c h had be e n w e t -
o xi d i z e d w i t h H N0 3 at ' l o w - t e mp e r at u r e ' ( Mi d d l e t o n & St u c k e y , 1 9 5 4 ) .
The d e t e r mi n at i o n o f t he u p t ak e o f c al c i u m by t he l e af w as bas e d o n me as u r e -
me n t s bo t h o f t he r e d u c t i o n o f Ca c o n c e n t r at i o n i n s o l u t i o n an d o f t he i n c r e as e i n
Ca c o n t e n t o f t he l e av e s . Cl o s e ag r e e me n t w as f o u n d be t w e e n Ca l o s s f r o m s o l u t i o n
an d Ca g ai n by l e av e s , an d t he r e w as n o e v i d e n c e o f me as u r abl e l o s s o f Ca by
r e t e n t i o n o n t he s u r f ac e s o f an y ap p ar at u s u s e d . The s e r e s u l t s w e r e c o n f i r me d by
r ad i o ac t i v e as s ay u s i n g Ca- 4 7 as a t r ac e r . In p r e p ar i n g i n f u s i o n s w i t h n at u r al
w at e r s , t he amo u n t s o f Ca l o s t by p r e c i p i t at i o n as c ar bo n at e w e r e n e g l i g i bl e w he n
t he w at e r s w e r e he at e d r ap i d l y t o bo i l i n g p o i n t an d u s e d i mme d i at e l y .
*
The av e r ag e c o n s u mp t i o n o f t e a i n t he Un i t e d K i n g d o m be t w e e n 1 9 5 6 an d 1 9 6 5 w as
1 1 - 3 g . p e r p e r s o n p e r d ay ( Nat i o n al F o o d Su r v e y Co mmi t t e e , 1 9 6 7 ) . Si n c e t he p r e s e n t s t u d i e s
p r o v i d e i n f o r mat i o n o n t he amo u n t s o f mat e r i al e xt r ac t e d f r o m 1 5 g . l e af , a u s e f u l g u i d e t o
t he av e r ag e d ai l y i n t ak e o f t he d i f f e r e n t t e a c o mp o n e n t s may be o bt ai n e d by mu l t i p l y i n g
t he amo u n t s r e p o r t e d he r e by a f ac t o r o f 0 7 5 .
r - 2
3
W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
The p u r e me t al an d al l o y p l at e s u s e d i n t e s t i n g t he c o r r o s i v e p o w e r o f i n f u s i o n s
w e r e o bt ai n e d f r o m v ar i o u s me t al w ar e man u f ac t u r e r s . B e f o r e u s e , t he p l at e s w e r e
r u bbe d d o w n f i r s t w i t h 2 4 0 g r ad e an d t he n w i t h 4 0 0 g r ad e e me r y p ap e r , an d
w as he d t ho r o u g hl y i n a s o l u t i o n o f d e t e r g e n t . The ar e a o f me t al s u r f ac e i mme r s e d
d u r i n g t he p r e p ar at i o n o f 1 l i t r e o f i n f u s i o n w as ap p r o xi mat e l y 5 0 0 c m2 .
Al u mi n i u m d e t e r mi n at i o n s w e r e mad e c o l o r i me t r i c al l y u s i n g t he au r i n t r i -
c ar bo xy l at e me t ho d ( San d e l l , 1 9 5 9 ) . P ho s p hat e w as d e t e r mi n e d by t he mo l y b-
d e n u m- bl u e c o l o r i me t r i c me t ho d , an d t o t al n i t r o g e n by t he K j e l d ahl me t ho d ,
f o l l o w i n g , i n bo t h c as e s , t he p r o c e d u r e s d e s c r i be d by P e ar s o n ( 1 9 6 2 ) .
Or g an i c
c o mp o n e n t s
P o l y p he n o l f r ac t i o n s w e r e o bt ai n e d by e t hy l ac e t at e e xt r ac t i o n an d l e ad s al t
p r e c i p i t at i o n ( Ro be r t s , Car t w r i g ht & Ol d s c ho o l , 1 9 5 7 ; Vu at az & B r an d e n bu r g e r ,
1 9 6 1 ) , an d w e r e an al y s e d by t w o - d i me n s i o n al c hr o mat o g r ap hy ( Ro be r t s e t al . 1 9 5 7 ) .
' Tan n i n ' w as d e t e r mi n e d by t he Lo w e n t hal me t ho d ( J ac o bs , 1 9 5 8 ) , c af f e i n e
s p e c t r o p ho t o me t r i c al l y ( P o l z e l l a, 1 9 6 1 ) , an d p hy t i c ac i d by t he me t ho d o f Mc Can c e
& Wi d d o w s o n ( 1 9 3 5 ) . The p e c t i n i c ac i d s w e r e c o n v e r t e d t o p e c t i c ac i d w hi c h w as
d e t e r mi n e d g r av i me t r i c al l y as t he Ca s al t ( Ca c o n t e n t ap p r o x. 7 5
% ) ,
f o l l o w i n g
c l o s e l y t he p r o c e d u r e r e c o mme n d e d by K e r t e s z ( 1 9 5 1 ) . The me t ho d u s e d f o r o xal at e
f o l l o w e d t he p r o c e d u r e d e s c r i be d by B ak e r ( 1 9 5 2 ) e xc e p t t hat c al c i u m s al t p r e c i p i -
t at i o n f r o m t he i n f u s i o n , w hi c h l e d t o p e r s i s t e n t c o n t ami n at i o n o f t he o xal at e , w as
r e p l ac e d by e t he r e xt r ac t i o n . Thu s , af t e r p r o t e i n r e mo v al , t he i n f u s i o n s w e r e
ac i d i f i e d t o 1 N s t r e n g t h w i t h H C1 an d c o n t i n u o u s l y e xt r ac t e d w i t h e t he r f o r 4 0 hr .
i n s u bd u e d l i g ht i n g . The e xt r ac t i o n e f f i c i e n c y , w hi c h w as mo n i t o r e d by r ad i o ac t i v e
as s ay o f ad d e d C- 1 4 l abe l l e d o xal i c ac i d , w as be t w e e n 8 5 an d 9 0 % .
RESULTS
The u p t ak e o f c al c i u m by t e a l e af
Me as u r e me n t s o f t he c al c i u m c o n t e n t o f al l e i g ht t e a s amp l e s g av e a me an v al u e
o f 5 5 ( r an g e 3 8 - 7 5 ) mg . Ca/ 1 5 g . d r y l e af . Di s t i l l e d w at e r i n f u s i o n s o f t he s e t e as
c o n t ai n e d be t w e e n 4 - 2 an d 5 - 8 mg . Ca/ l . s ho w i n g an e xt r ac t i o n o f o n l y 6 - 5 - 1 2 - 0
%
o f t he l e af c al c i u m. Wi t h i n f u s i o n s p r e p ar e d w i t h c al c i u m c hl o r i d e s o l u t i o n s c o n -
t ai n i n g 1 0 0 mg . Ca/ l . i t w as f o u n d t hat i n e ac h c as e t he r e w as a n e t u p t ak e o f
c al c i u m by t he l e af amo u n t i n g t o be t w e e n 1 6 an d 3 9 ( me an 3 0 ) mg . Ca/ 1 5 g . d r y
l e af , t he u p t ak e v al u e be i n g c har ac t e r i s t i c o f t he t e a s amp l e . Ev i d e n c e t hat t he s e
n e t u p t ak e f i g u r e s w e r e n o t s u bs t an t i al l y s mal l e r t han t he t o t al c al c i u m u p t ak e
v al u e s w as o bt ai n e d by ac t i v i t y me as u r e me n t s o f Ca- 4 7 , ad d e d t o t he c al c i u m
s o l u t i o n s p r i o r t o p r e p ar i n g t he i n f u s i o n s . Thu s , i t w as f o u n d t hat t he f r ac t i o n al
l o s s o f Ca- 4 7 ac t i v i t y f r o m t he s o l u t i o n s w as c l o s e l y s i mi l ar i n mag n i t u d e t o t he
f r ac t i o n al l o s s o f s t abl e c al c i u m. Mo r e d e t ai l e d s t u d i e s o f t he p at t e r n o f c al c i u m
u p t ak e w e r e mad e w i t h f o u r t e a t y p e s ( A, C, E, H ) u s i n g s o l u t i o n s w i t h c al c i u m
c o n t e n t s r an g i n g f r o m 0 t o 1 5 0 mg . Ca/ l . In e ac h i t w as f o u n d t hat t he u p t ak e c u r v e
r o s e s t e e p l y o v e r t he l o w e r hal f o f t he r an g e an d f l at t e n e d o u t w he n t he s o l u t i o n
c al c i u m w as at abo u t 1 0 0 mg . Ca/ l . A t y p i c al s e t o f r e s u l t s i s s ho w n i n F i g . 1 .
4
Te a
i n f u s i o n s
an d w at e r har d n e s s 5
Te s t s w i t h s o l u t i o n s o f c al c i u m at 1 0 0 mg . Ca/ l . as s u l p hat e an d n i t r at e s ho w e d
c al c i u m u p t ak e v al u e s w hi c h w e r e n o t s i g n i f i c an t l y d i f f e r e n t f r o m t he c o r r e s p o n d i n g
v al u e s o bt ai n e d w i t h c al c i u m c hl o r i d e s o l u t i o n s . Cal c i u m u p t ak e me as u r e me n t s
w e r e al s o mad e u s i n g s amp l e s o f t hr e e n at u r al d r i n k i n g - w at e r s i n w hi c h t he bi -
c ar bo n at e c o n t e n t w as r o u g hl y e q u al i n e q u i v al e n t s t o t he c al c i u m c o n t e n t ( Io n
c o n t e n t o f w at e r s , mg / l . : ( 1 ) Ca, 1 5 ; Mg , 3 3 ; Na, 3 4 ; H CO3 , 4 1 : ( 2 ) Ca, 6 6 ; Mg , 3 4 ;
Na, 2 0 ; H CO3 , 2 2 2 : ( 3 ) Ca, 8 3 ; Mg , 5 4 ; Na, 6 0 ; H CO3 , 2 4 0 ) . F o r e ac h w at e r i t w as
f o u n d t hat t he u p t ak e w as i n g o o d ag r e e me n t w i t h t he v al u e e xp e c t e d o n t he bas i s
o f t he r e s u l t s f o r t he c al c i u m c hl o r i d e s o l u t i o n s . The p H v al u e s o f t he i n f u s i o n s
4 0
3 0 0
E /
- 2 0
EI
1 0
z
0
5 0 1 0 0 1 5 0
Co n c e n t r at i o n o f c al c i u m i n w at e r ( mg . / I. )
- 1 0
F i g . 1 . The r e l at i o n s hi p be t w e e n t he n e t amo u n t o f c al c i u m t ak e n u p by t e a l e af d u r i n g
t he p r e p ar at i o n o f i n f u s i o n s an d t he i n i t i al c o n c e n t r at i o n o f c al c i u m i n t he e xt r ac t i n g
w at e r ( t e a s amp l e A) . The maj o r p ar t o f t he an al y t i c al e r r o r i n c al c i u m d e t e r mi n at i o n
w as d u e t o t he r an d o m e r r o r o f t he f l ame p ho t o me t r i c me t ho d u s e d ; e r r o r s s ho w n
( 2 o - ) ar e t he t o t al p ho t o me t r i c e r r o r s i n t r o d u c e d i n t o t he c al c u l at e d c al c i u m u p t ak e
v al u e s .
p r e p ar e d w i t h t he n e u t r al s al t s o l u t i o n s w e r e e s s e n t i al l y d e t e r mi n e d by t he bu f f e r -
i n g ac t i o n o f t he c o mp o n e n t s e xt r ac t e d f r o m t he l e af , an d f e l l w i t hi n t he r an g e p H
4 - 4 - 4 9 . The p H v al u e s o f t he i n f u s i o n s w i t h n at u r al w at e r s amp l e s 2 an d 3 w e r e
c l o s e t o 5 - 3 . F u r t he r t e s t s w e r e c ar r i e d o u t u s i n g ac e t at e an d c o l l i d i n e bu f f e r s ( at
0 0 5 M) t o g i v e i n f u s i o n s i n w hi c h t he p H r e ac he d 6 - 5 bu t n o s i g n i f i c an t e f f e c t o f p H
o n t he c al c i u m u p t ak e w as o bs e r v e d . Thu s , i n g e n e r al i t ap p e ar e d t hat t he p at t e r n
o f c al c i u m u p t ak e ( F i g . 1 ) w as n o t s i g n i f i c an t l y af f e c t e d by t he p r e s e n c e o f t he
o t he r p r i n c i p al i o n s c o mmo n l y p r e s e n t i n d r i n k i n g - w at e r s ( Tay l o r , 1 9 5 8 ) , o r by
t he d i f f e r e n c e s i n p H n o r mal l y e n c o u n t e r e d i n t e a i n f u s i o n s .
St u d i e s o f i n f u s i o n s p r e p ar e d w i t h s o d i u m c hl o r i d e an d mag n e s i u m c hl o r i d e
s o l u t i o n s s ho w e d t hat t he r e w as n o an al o g o u s u p t ak e o f s o d i u m o r mag n e s i u m by
5
W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
t he l e af . In f ac t t he p r e s e n c e o f Mg i o n s i n t he s t ar t i n g s o l u t i o n c au s e d an ad d i t i o n al
r e l e as e o f t he mag n e s i u m p r e s e n t i n t he l e af . Thu s , s o l u t i o n s o f mag n e s i u m c hl o r i d e
at 5 0 mg . Mg / I. e xt r ac t e d abo u t 8 0
%
o f t he l e af mag n e s i u m c o mp ar e d w i t h abo u t
6 0
%
e xt r ac t e d i n d i s t i l l e d w at e r i n f u s i o n s .
F u r t he r e xp e r i me n t s w i t h c al c i u m c hl o r i d e s o l u t i o n s s ho w e d t hat t he c al c i u m
u p t ak e p r o c e s s w as f ai r l y r ap i d an d e s s e n t i al l y c o mp l e t e w i t hi n 2 mi n . Te s t s w e r e
al s o mad e o n l e av e s r e c o v e r e d f r o m i n f u s i o n s af t e r a 6 mi n . p e r i o d . Thu s , i n e xp e r i -
me n t s i n w hi c h l e av e s , s e p ar at e d f r o m i n f u s i o n s w i t h c al c i u m c hl o r i d e s o l u t i o n s ,
w e r e s u bj e c t e d t o s e v e r al ad d i t i o n al e xt r ac t i o n s w i t h d i s t i l l e d w at e r , i t w as f o u n d
t hat o n l y a s mal l p e r c e n t ag e o f t he c al c i u m t ak e n u p by t he l e af w as e xt r ac t e d i n
e ac h s u c c e s s i v e i n f u s i o n w i t h d i s t i l l e d w at e r . It w as al s o f o u n d t hat l e av e s w hi c h
had be e n s e p ar at e d f r o m i n f u s i o n s w i t h d i s t i l l e d w at e r s ho w e d n o ap p r e c i abl e u p -
t ak e o f c al c i u m i n s u bs e q u e n t i n f u s i o n s w i t h c al c i u m c hl o r i d e s o l u t i o n s . Thu s , i t
ap p e ar e d t hat t he l e af c o mp o n e n t r e s p o n s i bl e f o r t he u p t ak e an d bi n d i n g o f c al c i u m
w as e i t he r al t e r e d o r r e mo v e d d u r i n g t he p r e p ar at i o n o f a d i s t i l l e d w at e r i n f u s i o n .
Co mp ar at i v e s t u d i e s w e r e t he r e f o r e mad e o f t he e xt r ac t i o n o f v ar i o u s c o mp o n e n t s
o f t he t e a l e af by w at e r s o f d i f f e r e n t c al c i u m c o n t e n t .
The c o mp o s i t i o n o f t e a i n f u s i o n s
P r e l i mi n ar y me as u r e me n t s o f t he t o t al s o l i d s p r e s e n t i n t e a i n f u s i o n s s ho w e d t hat
abo u t o n e - t hi r d o f t he l e af mat e r i al had be e n e xt r ac t e d ( Tabl e 1 ) , an d t hat t he r e
w as n o l ar g e d i f f e r e n c e be t w e e n t he amo u n t s e xt r ac t e d i n d i s t i l l e d w at e r an d
Tabl e 1 . G e n e r al c o mp o s i t i o n o f t e a i n f u s i o n s
Av e r ag e c o n c e n t r at i o n i n i n f u s i o n s o f
1 5 g . l e af p e r l i t r e o f d i s t i l l e d w at e r ( g . / l . )
( F i g u r e s i n br ac k e t s r e p r e s e n t n u mbe r o f
s amp l e s s t u d i e d )
Co mp o n e n t P r e s e n t s t u d i e s P r e v i o u s s t u d i e s
To t al s o l i d s 5 . 1 0 ( 8 )
In o r g an i c c at i o n s 0 2 3 ( 8 ) 0 - 2 8 ( 5 ) a
P o l y p he n o l s 2 . 3 0 ( 2 ) 2 . 5 0 ( l ) b
Caf f e i n e 0 5 3 ( 3 ) 0 5 1 ( 4 ) c
P r o t e i n an d ami n o ac i d s d 0 - 8 5 ( 3 ) 1 . 0 0 ( 5 ) a
Mi s c e l l an e o u s 1 . 1 5 ( 2 )
c o mp o n e n t c o n s i s t i n g
mai n l y o f s u g ar s ( by
d i f f e r e n c e )
a, Mc Can c e & Wi d d o w s o n , 1 9 5 6 ; b, Ro be r t s e t al . 1 9 5 7 ; c , Smi t h & Re e s , 1 9 6 3 ; d , K j e l d ahl
N ( c o r r e c t e d f o r c af f e i n e N) x 6 - 2 5 .
c al c i u m- l o ad e d i n f u s i o n s . A mo r e d e t ai l e d s e t o f me as u r e me n t s ( Samp l e s A, C)
i n d i c at e d t hat t he p r e s e n c e o f c al c i u m i n t he s t ar t i n g s o l u t i o n ( 1 0 0 mg . Ca/ I. )
r e d u c e d t he amo u n t o f mat e r i al e xt r ac t e d by be t w e e n 2 an d 3
% .
The r e s u l t s o f
s t u d i e s o f i n d i v i d u al c o mp o n e n t s
ar e n o w s u mmar i z e d .
6
Te a i n f u s i o n s an d w at e r har d n e s s
Me t al i o n s
Me as u r e me n t s w e r e mad e o f t he me t al i o n c o n t e n t o f al l e i g ht t e a s amp l e s , an d
al s o o f t he amo u n t s o f t he s e i o n s e xt r ac t e d i n d i s t i l l e d w at e r i n f u s i o n s . The r e s u l t s
o f t he s e me as u r e me n t s ar e s u mmar i z e d i n Tabl e 2 . At t e n t i o n i s d r aw n t o t he
r e l at i v e l y hi g h l e v e l s o f al u mi n i u m, man g an e s e an d z i n c f o u n d i n t he s amp l e s .
An al y s e s o f i n f u s i o n s p r e p ar e d w i t h s o l u t i o n s o f c al c i u m at 1 0 0 mg . Ca/ l . s ho w e d
t hat , e xc e p t f o r an i n c r e as e by abo u t 2 5
%
i n t he amo u n t o f mag n e s i u m e xt r ac t e d ,
t he e xt r ac t i o n o f t he me t al s l i s t e d i n Tabl e 2 w as u n af f e c t e d by t he p r e s e n c e o f
c al c i u m i n t he s t ar t i n g s o l u t i o n . As n o t e d abo v e , t he e xt r ac t i o n o f l e af mag n e s i u m
w as al s o i n c r e as e d t o a s i mi l ar e xt e n t by t he p r e s e n c e o f Mg i o n s i n t he s t ar t i n g
Tabl e 2 . Me t al i o n s d e t e c t e d i n t e a l e af an d i n d i s t i l l e d w at e r
i n f u s i o n s ( av e r ag e o f e i g ht t e a s amp l e s )
Amo u n t i n d r y l e af Amo u n t i n
( mg . / 1 5 g . ) i n f u s i o n
A
- 1 a ( mg . / l . )
El e me n t Mi n . Max. Me an Me an
Na 5 1 0 8 . 0 7 . 2
Mg 3 6 5 1 4 4 2 5
Al a 3 1 4 9 - 8 2 . 8
K 1 3 5 3 8 0 2 2 5 1 8 2
Ca 3 8 7 5 5 5 5 - 1
Mn 5 2 3 1 8 5 . 4
F e 2 5 3 . 5 < 0 2 5
Z n 0 - 5 1 5 4 . 0 2 . 6
Sr 0 1 8 0 6 5 0 . 3 7 0 0 6
a, De t e r mi n at i o n s o f Al w e r e mad e o n o n l y 5 t e a s amp l e s .
s o l u t i o n . A mo r e d e t ai l e d s t u d y , u s i n g v ar i o u s i o n i c c o n c e n t r at i o n s o f c al c i u m o r
mag n e s i u m i n t he s t ar t i n g s o l u t i o n s , s ho w e d t hat t he ad d i t i o n al r e l e as e o f l e af
mag n e s i u m c au s e d by Ca i o n s w as c l o s e l y s i mi l ar i n mag n i t u d e t o t hat c au s e d by
t he s ame i o n i c c o n c e n t r at i o n s o f Mg i o n s . It t he r e f o r e ap p e ar e d l i k e l y t hat t he
i n f l u e n c e o f Ca i o n s o n mag n e s i u m e xt r ac t i o n w as d u e t o an i o n i c c o n c e n t r at i o n
e f f e c t o n t he s o l u bi l i t y o f t he mag n e s i u m c o mp o n e n t p r e s e n t i n t he l e af , al t ho u g h
t he p o s s i bi l i t y t hat s o me c al c i u m u p t ak e o c c u r s by i o n e xc han g e w i t h l e af mag -
n e s i u m w as n o t f u l l y e xc l u d e d .
To o bt ai n i n f o r mat i o n o n t he d e g r e e t o w hi c h t he t r ac e me t al c o n t e n t o f t e a
i n f u s i o n s may be i n c r e as e d by c o n t ami n at i o n f r o m me t al w ar e c u r r e n t l y o r f o r me r l y
u s e d i n t he i r p r e p ar at i o n , a s e r i e s o f me as u r e me n t s w as mad e o f t he me t al c o n t e n t
o f d i s t i l l e d w at e r i n f u s i o n s p r e p ar e d
i n t he p r e s e n c e
o f v ar i o u s p u r e me t al o r al l o y
p l at e s . No e v i d e n c e w as f o u n d o f me t al c o n t ami n at i o n at l e v e l s abo v e 1 mg . me t al / l .
i n f u s i o n f r o m s amp l e s o f al u mi n i u m,
s t ai n l e s s
s t e e l , mo d e r n p e w t e r , s i l v e r , o r
c hr o mi u m- p l at e d n i c k e l - p l at e d c o p p e r . Me t al p i c k - u p by i n f u s i o n s w as o bs e r v e d
f r o m s amp l e s o f c o p p e r ( 8 mg . Cu / l . i n f u s i o n ) ,
n i c k e l
( 2 0 mg . Ni / l . ) , n i c k e l - s i l v e r
( [ 1 mg . Z n + 2 mg . Ni + 7 mg . Cu ] / l . ) ,
an d br as s ( [ 3 mg .
Z n
+ 3 mg . Cu ] / 1 . ) . In f u s i o n s
w e r e al s o p r e p ar e d w i t h c al c i u m- l o ad e d s o l u t i o n s i n t he p r e s e n c e o f n i c k e l an d
7
W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
c o p p e r p l at e s bu t n o e v i d e n c e w as f o u n d o f an y mar k e d i n f l u e n c e o f t he c al c i u m
o n t he c o r r o s i v e p o w e r o f t he i n f u s i o n s . F u r t he r mo r e , t he u p t ak e o f c al c i u m
by t he l e av e s w as u n af f e c t e d by t he p r e s e n c e o f t he me t al p l at e s .
Te s t s o n d i s t i l l e d w at e r i n f u s i o n s p r e p ar e d i n an o l d , l e ad - c o n t ai n i n g , p e w t e r
t e ap o t ( 6
%
P b) s ho w e d t he p e r s i s t e n t o c c u r r e n c e o f a c o mp ar at i v e l y hi g h p i c k - u p
o f P b ( 2 - 4 mg . P b/ l . i n f u s i o n ) w i t h l i t t l e t e n d e n c y f o r a p r o t e c t i v e c o at i n g t o f o r m
i n s i d e t he p o t . In f u s i o n s p r e p ar e d w i t h c al c i u m- l o ad e d s o l u t i o n s t e n d e d t o l e av e
a f i l m o n t he me t al s u r f ac e an d i n t he s e i n f u s i o n s t he l e ad c o n t e n t w as r e d u c e d t o
abo u t 1 mg . P b/ l .
P o l y p he n o l s
A p r e l i mi n ar y i n v e s t i g at i o n o f t he ' t an n i n ' c o mp o n e n t ( r e d u c i n g p o l y p he n o l s )
o f i n f u s i o n s by t he L6 w e n t hal me t ho d i n d i c at e d t hat t he p r e s e n c e o f c al c i u m i n t he
e xt r ac t i n g l i q u i d had n o i n f l u e n c e o n t he e xt r ac t i o n o f t hi s c o mp o n e n t . The p o l y -
p he n o l i c f r ac t i o n s p r e s e n t i n d i s t i l l e d w at e r an d c al c i u m- l o ad e d i n f u s i o n s o f t w o
t e a s amp l e s ( A, C) w e r e s e p ar at e d an d an al y s e d by t w o - d i me n s i o n al p ap e r c hr o -
mat o g r ap hy . De t ai l e d e xami n at i o n o f t he ban d s an d s p o t s o n t he c hr o mat o g r ams ,
w hi c h d i s p l ay e d t he maj o r t he ar u bi g i n an d t he af l av i n c o mp o n e n t s t o g e t he r w i t h
a w i d e r an g e o f f l av o n o i d an d o t he r p o l y p he n o l s , g av e n o e v i d e n c e t hat c al c i u m i n
t he e xt r ac t i n g l i q u i d had a mar k e d e f f e c t o n t he e xt r ac t i o n o f an y o f t he s e c o m-
p o u n d s . The t o t al w e i g ht s o f p o l y p he n o l i c s u bs t an c e s i s o l at e d f r o m t he i n f u s i o n s
w e r e 2 - 1 0 g . ( Samp l e A) an d 2 - 5 0 g . ( Samp l e C) .
To t al n i t r o g e n an d c af f e i n e
Me as u r e me n t s o f t he t o t al N c o n t e n t o f t he s o l i d s r e c o v e r e d f r o m i n f u s i o n s
p r e p ar e d bo t h w i t h d i s t i l l e d w at e r an d c al c i u m c hl o r i d e s o l u t i o n s ( 1 0 0 mg . Ca/ l . )
i n d i c at e d t hat t he p r e s e n c e o f c al c i u m had n o ap p r e c i abl e i n f l u e n c e o n t he e xt r ac -
t i o n o f N- c o n t ai n i n g c o mp o n e n t s ( mai n l y p r o t e i n , ami n o ac i d s an d c af f e i n e ) . Thi s
i n d i c at i o n w as c o n f i r me d d i r e c t l y i n r e s p e c t o f t he c af f e i n e c o n t e n t o f i n f u s i o n s o f
t hr e e t e as ( c af f e i n e c o n t e n t o f i n f u s i o n s o f Samp l e s A, C, E: 0 - 5 4 , 0 5 8 an d 0 4 4 g . / l . ) .
P e c t i n i c ac i d s
In p r e l i mi n ar y t e s t s o n t he s o l u bi l i t y o f t he p e c t i c mat e r i al p r e s e n t i n t e a, ho t
e xt r ac t s o f t w o t e as ( A, C) w e r e mad e by s t i r r i n g s amp l e s i n d i s t i l l e d w at e r o n t he
bo i l i n g w at e r bat h f o r an 8 - hr . p e r i o d . The w e i g ht s o f p e c t i c ac i d r e c o v e r e d f r o m
t he s e e xt r ac t s amo u n t e d t o 1 - 6 an d 2 - 6
%
o f t he d r y l e af w e i g ht . Whe n s i mi l ar
e xt r ac t s w e r e mad e at r o o m t e mp e r at u r e t he r e w as a t e n f o l d r e d u c t i o n i n t he
amo u n t s o f p e c t i c mat e r i al e xt r ac t e d .
The d e t e r mi n at i o n o f t he p e c t i c mat e r i al p r e s e n t i n n o r mal 6 mi n . i n f u s i o n s w i t h
d i s t i l l e d w at e r g av e v al u e s f o r t hr e e t e as ( A, C, E) o f 3 3 , 3 4 an d 1 8 mg . p e c t i c
ac i d / l . Whe n t he i n f u s i o n s w e r e p r e p ar e d w i t h s o l u t i o n s c o n t ai n i n g c al c i u m at
1 0 0 mg . Ca/ l . , t he p e c t i c c o n t e n t w as s u bs t an t i al l y r e d u c e d t o v al u e s o f be t w e e n
5 an d 1 0 mg . p e c t i c ac i d / l . Co n t r o l t e s t s s ho w e d t hat t he p r e s e n c e o f c al c i u m i n t he
i n f u s i o n d i d n o t i n t e r f e r e w i t h t he s e d e t e r mi n at i o n s . It ap p e ar e d t he r e f o r e t hat
8
Te a i n f u s i o n s an d w at e r har d n e s s 9
t he p r e c i p i t at i o n o f l o w - e s t e r c al c i u m p e c t i n at e s w i t hi n t he l e af w o u l d ac c o u n t f o r
s o me o f t he c al c i u m u p t ak e f r o m s o l u t i o n , bu t t he q u an t i t y o f c al c i u m i n v o l v e d i n
t hi s p r o c e s s w o u l d amo u n t t o o n l y 1 - 2 mg . Ca/ 1 5 g . d r y l e af .
Oxal i c ac i d
De t e r mi n at i o n s o f t he o xal at e c o n t e n t o f d i s t i l l e d w at e r i n f u s i o n s o f s i x t e as
( A, B , C, D, E, F ) g av e v al u e s r an g i n g f r o m 6 2 t o 9 8 ( me an 8 2 5 ) mg . o xal at e / l .
The c o r r e s p o n d i n g v al u e s f o r i n f u s i o n s w i t h c al c i u m c hl o r i d e s o l u t i o n s
( 1 0 0
mg .
Ca/ l . ) r an g e d f r o m 3 9 t o 4 8 ( me an 4 3 5 ) mg . o xal at e / l . An al y s i s o f t he r e s u l t s s ho w e d
1 0 0
r -
Z -
E
C
0
C
.
4 )
' 4
. v
x
0
0
C
0
. 2
' 4
4 c
C
0
u
5 0
5 0 1 0 0 1 5 0
Co n c e n t r at i o n o f c al c i u m i n w at e r ( mg . / I. )
F i g . 2 . The r e l at i o n s hi p be t w e e n t he amo u n t o f o xal at e i o n i n t e a i n f u s i o n s an d t he
i n i t i al c o n c e n t r at i o n o f c al c i u m i n t he e xt r ac t i n g w at e r ( t e a s amp l e A) . The o v e r al l
an al y t i c al e r r o r s i n t he o xal at e v al u e s ar e e s t i mat e d t o be l e s s t han + 5 % o f t he
p l o t t e d v al u e s .
t hat o n t he av e r ag e t he o xal at e c o n t e n t o f t he c al c i u m- l o ad e d i n f u s i o n s w as 5 3 - 5
%
t hat o f t he d i s t i l l e d w at e r i n f u s i o n s . Co n t r o l t e s t s s ho w e d t hat t he p r e s e n c e o f
c al c i u m i n t he i n f u s i o n s d i d n o t i n t e r f e r e w i t h t he o xal at e d e t e r mi n at i o n s an d
g av e n o e v i d e n c e t hat c al c i u m o xal at e w as p r e c i p i t at e d f r o m c al c i u m- l o ad e d
i n f u s i o n s o n t o t he s u r f ac e s o f an y ap p ar at u s u s e d . F u r t he r an al y s i s o f t he r e s u l t s
i n r e l at i o n t o t he v al u e s f o u n d f o r c al c i u m u p t ak e by t he l e af s ho w e d t hat t he
d e p o s i t i o n o f c al c i u m o xal at e i n t he l e af w o u l d ac c o u n t f o r be t w e e n 4 2 an d 8 2
( me an 6 1 ) %
o f t he c al c i u m u p t ak e v al u e s . A s e r i e s o f me as u r e me n t s o f t he o xal at e
i n i n f u s i o n s p r e p ar e d w i t h d i f f e r e n t amo u n t s o f c al c i u m i n t he e xt r ac t i n g l i q u i d
s ho w e d t hat t he r e w as a p r o g r e s s i v e d e c r e as e i n t he amo u n t o f o xal at e e xt r ac t e d
as t he c al c i u m c o n c e n t r at i o n w as i n c r e as e d f r o m 0 t o 1 5 0
mg . Ca/ l . ( F i g . 2 ) .
1 0 W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
Var i o u s t e s t s w e r e c ar r i e d o u t t o d i s c o v e r t he f at e o f t he o xal at e w he n mi l k w as
ad mi xe d w i t h bo t h d i s t i l l e d w at e r i n f u s i o n s an d c al c i u m- l o ad e d i n f u s i o n s i n t he
r at i o o f mi l k : i n f u s i o n = 1 : 5 . F o l l o w i n g d o me s t i c p r ac t i c e , t he mi xt u r e s w e r e
p r e p ar e d e i t he r by p o u r i n g t he i n f u s i o n s o n t o t he mi l k , o r v i c e v e r s a, an d s t i r r i n g
br i e f l y . The mi xt u r e s w e r e t he n al l o w e d t o s t an d f o r 1 0 mi n . De t e r mi n at i o n o f t he
o xal at e c o n t e n t o f t he mi xt u r e s s ho w e d t hat mo r e t han 9 0
%
o f t he o xal at e r e mai n e d
w i t hi n t he bo d y o f t he mi xt u r e . F u r t he r c he c k s o n t he o xal at e c o n t e n t bo t h o f
t he u n d i s t u r be d bo t t o m l ay e r s o f mi xt u r e s he l d i n t e a- c u p s f o r 1 0 mi n . , an d al s o o f
an y mat e r i al d e p o s i t e d o n t he s u r f ac e s o f t he c u p s , c o n f i r me d t hat l e s s t han 1 0
%
o f t o t al o xal at e had s e t t l e d o u t o f t he mi xt u r e s .
P hy t i c ac i d an d p ho s p hat e
Us i n g a me t ho d c ap abl e o f d e t e c t i n g p hy t at e i f p r e s e n t at c o n c e n t r at i o n s abo v e
5 mg . / 1 5 g . l e af , n o me as u r abl e p hy t at e w as f o u n d i n 0 5 N- H CI e xt r ac t s o f f o u r
t e as ( A, C, E, G ) . Thi s r e s u l t i n d i c at e s t hat , i n t he p r o c e s s o f c al c i u m u p t ak e by t he
l e af i n c al c i u m- l o ad e d i n f u s i o n s , d e p o s i t i o n o f c al c i u m p hy t at e w o u l d n o t ac c o u n t
f o r mo r e t han 1 mg . Ca/ 1 5 g . d r y l e af . De t e r mi n at i o n s o f t he p ho s p hat e c o n t e n t o f
d i s t i l l e d w at e r an d c al c i u m- l o ad e d i n f u s i o n s c ar r i e d o u t f o r o n e t e a s amp l e ( A)
s ho w e d t hat t he p r e s e n c e o f c al c i u m w as w i t ho u t i n f l u e n c e o n t he t o t al p ho s p hat e
e xt r ac t e d ( p ho s p hat e c o n t e n t o f i n f u s i o n : 2 5 - 0 mg . P / 1 . ) .
DISCUSSION
The ' har d n e s s ' o f d r i n k i n g w at e r s i s d u e t o t he i r c o n t e n t o f c al c i u m an d mag -
n e s i u m i o n s . In t he maj o r i t y o f w at e r s i n t he Un i t e d K i n g d o m c al c i u m i s t he
p r i n c i p al c o mp o n e n t o f ' har d n e s s ' , t he c al c i u m c o n t e n t r an g i n g f r o m l e s s t han
1 mg . Ca p e r l i t r e i n v e r y s o f t w at e r s t o abo u t 1 5 0 mg . Ca p e r l i t r e i n t he har d e s t
o f w at e r s n o r mal l y s u p p l i e d ( Sk e at , 1 9 6 1 ) .
An
i mp o r t an t
f r ac t i o n o f t he c al c i u m
i n mo s t w at e r s i s p r e s e n t as c al c i u m bi c ar bo n at e ( ' t e mp o r ar y har d n e s s ' ) , an d
d u r i n g t he bo i l i n g o f w at e r s f o r t he p r e p ar at i o n
o f t e a i n f u s i o n s s o me l o s s o f
c al c i u m f r o m s o l u t i o n , by d e p o s i t i o n as c ar bo n at e ,
w i l l
g e n e r al l y o c c u r f o r w at e r s
c o n t ai n i n g mo r e t han abo u t 1 0 mg . c al c i u m p e r l i t r e . An i n d i c at i o n o f t he e xt e n t
o f t hi s l o s s i s p r o v i d e d by H o l l i n s ( 1 9 6 5 )
w ho f o u n d t hat ,
w he n s o l u t i o n s c o n t ai n i n g
e q u i v al e n t amo u n t s o f c al c i u m an d bi c ar bo n at e i o n s w e r e he at e d t o bo i l i n g o v e r
a 5 mi n . p e r i o d an d bo i l e d f o r a f u r t he r 3 mi n . ,
be t w e e n 1 0 an d 2 0
%
o f t he c al c i u m
w as d e p o s i t e d f r o m s o l u t i o n s w hi c h had s t ar t i n g
c o n c e n t r at i o n s o f 5 0 an d 1 0 0 mg .
c al c i u m p e r l i t r e . The p r e s e n t s t u d i e s s ho w t hat a f u r t he r s u bs t an t i al l o s s o f
c al c i u m o c c u r s f r o m d r i n k i n g - w at e r s d u r i n g t he p r e p ar at i o n
o f t e a i n f u s i o n s d u e
t o c al c i u m u p t ak e by t he t e a l e af . The g e n e r al p at t e r n o f t hi s
u p t ak e o v e r t he r an g e
o f c al c i u m c o n c e n t r at i o n n o r mal l y
f o u n d i n
d r i n k i n g - w at e r s
i s i n d i c at e d
by
t he
c u r v e i n F i g . 1 . As o u t l i n e d i n t he In t r o d u c t i o n ,
o u r
p r i mar y
c o n c e r n he r e i s w i t h
t he i n f l u e n c e o f t he c al c i u m o n t he c o mp o s i t i o n o f t he
i n f u s i o n s ,
an d f o r t he
p r e s e n t
p u r p o s e i t w i l l s u f f i c e t o n o t e t hat c al c i u m u p t ak e
w as s ho w n i n s i mi l ar
d e g r e e by
al l t he t e as e xami n e d an d t hat t he u p t ak e p r o c e s s
w as
e s s e n t i al l y
u n af f e c t e d
by
t he p r e s e n c e o f an y o f t he o t he r p r i n c i p al
i o n s n o r mal l y
f o u n d i n
d r i n k i n g - w at e r s .
Te a i n f u s i o n s an d w at e r har d n e s s
The r e s u l t s f o u n d o n t he c o mp o s i t i o n o f t he i n f u s i o n s w i l l n o w be d i s c u s s e d , p ar t i -
c u l ar l y i n r e l at i o n t o t he p r o bl e m o f t he as s o c i at i o n be t w e e n w at e r har d n e s s an d
mo r t al i t y .
Se v e r al au t ho r s ( Mo r r i s e t al . 1 9 6 1 ; Sc hr o e d e r , 1 9 6 6 ; Dav i e s , 1 9 6 2 ) hav e s u g g e s t e d
t hat t he ' w at e r f ac t o r ' i n c ar d i o v as c u l ar d i s e as e mi g ht be r e l at e d t o t he t r ac e
e l e me n t c o n t e n t o f d r i n k i n g - w at e r s bu t n o f i r m e v i d e n c e has y e t be e n p r o d u c e d t o
s u bs t an t i at e t hi s v i e w i n r e s p e c t o f an y s i n g l e e l e me n t . In t e r e s t i n t he i n t ak e o f
t r ac e e l e me n t s f r o m d r i n k i n g - w at e r s i s w e ak e n e d by t he f ac t t hat t he c o n t r i bu t i o n
o f t he s e e l e me n t s f r o m w at e r i s g e n e r al l y a s mal l f r ac t i o n o f t he t o t al d i e t ar y
i n t ak e . The p r e s e n t r e s u l t s u n d e r l i n e t hi s f ac t s p e c i f i c al l y f o r man g an e s e , z i n c , an d
al u mi n i u m s i n c e t he amo u n t s o f t he s e me t al s e xt r ac t e d f r o m t he t e a l e af i n t o t he
i n f u s i o n ar e hi g he r by an o r d e r o f mag n i t u d e t han t he c o r r e s p o n d i n g amo u n t s
mo s t f r e q u e n t l y f o u n d i n d r i n k i n g - w at e r s ( Sc hr o e d e r , B al as s a & Ti p t o n , 1 9 6 6 ;
H o l l i n s , 1 9 6 5 ; Camp be l l , Cas s , Cho l ak & K e ho e , 1 9 5 7 ) . The r e s u l t s o f o u r t e s t s o n
me t al p i c k - u p by i n f u s i o n s i n c o n t ac t w i t h v ar i o u s me t al l i c s u r f ac e s i n d i c at e t hat
t he me t al w ar e mo s t c o mmo n l y u s e d at t he p r e s e n t t i me f o r t he p r e p ar at i o n o f t e a
i n f u s i o n s d o e s n o t g i v e r i s e t o an y ap p r e c i abl e c o n t ami n at i o n o f t he i n f u s i o n ,
al t ho u g h p i c k - u p o f c o p p e r , n i c k e l , z i n c an d l e ad t o a l e v e l o f s e v e r al mi l l i g r ams
p e r l i t r e may be e xp e c t e d t o o c c u r i n i n f u s i o n s p r e p ar e d i n c e r t ai n t y p e s o f t e a-
p o t s man u f ac t u r e d at e ar l i e r t i me s . Of t he s e v ar i o u s t r ac e me t al s , l e ad c an be
s i n g l e d o u t as o n e c ar r y i n g a he al t h haz ar d at a c o mp ar at i v e l y l o w i n t ak e l e v e l
( Mo n i e r - Wi l hi ams , 1 9 4 9 ) , s o t hat an y c o n t i n u al u s e o f l e ad - p e w t e r v e s s e l s f o r
p r e p ar i n g t e a i n f u s i o n s s ho u l d be d i s c o u r ag e d . In t hi s c o n n e xi o n i t s ho u l d be
n o t e d t hat t he s p u n p e w t e r u s e d i n p r e s e n t - d ay c o o k i n g u t e n s i l s i s e s s e n t i al l y
l e ad - f r e e . Wi t h r e g ar d t o t he o t he r t r ac e me t al s , i t i s u n c e r t ai n ( Camp be l l e t al .
1 9 5 7 ; Un d e r w o o d , 1 9 6 2 ) w he t he r i n t ak e s o f man g an e s e , z i n c , al u mi n i u m, c o p p e r
o r n i c k e l hav e an y s i g n i f i c an t p hy s i o l o g i c al i n f l u e n c e at t he amo u n t s f o u n d f o r t e a
i n f u s i o n s al t ho u g h i t s e e ms p o s s i bl e t hat t he z i n c an d man g an e s e c o n t r i bu t i o n s
may be u s e f u l s u p p l e me n t s t o e s s e n t i al d i e t ar y r e q u i r e me n t s . The r e i s , ho w e v e r ,
n o e v i d e n c e f r o m t he p r e s e n t r e s u l t s o f an y s t r i k i n g r e l at i o n be t w e e n t he t r ac e
me t al c o mp o s i t i o n o f t he i n f u s i o n s an d t he har d n e s s o f t he e xt r ac t i n g w at e r . H ar d
w at e r s t e n d t o e xt r ac t mo r e mag n e s i u m f r o m t e a t han d o s o f t w at e r s , bu t t he
i n c r e me n t i n v o l v e d ( abo u t 1 0 mg . Mg / l . i n f u s i o n ) ap p e ar s t r i v i al w he n c o mp ar e d
w i t h t he n o r mal d ai l y i n t ak e o f mag n e s i u m ( abo u t 3 0 0 mg . ) f r o m o t he r d i e t ar y
s o u r c e s ( Wac k e r & Val l e e , 1 9 6 4 ) .
It i s al s o c l e ar f r o m o u r s t u d i e s t hat t he c al c i u m i n d r i n k i n g - w at e r s d o e s n o t
hav e an y n o t abl e i n f l u e n c e o n t he e xt r ac t i o n o f t he o t he r maj o r t e a c o mp o n e n t s
l i s t e d i n Tabl e 1 . Sp e c i al at t e n t i o n w as g i v e n t o t he c o mp l e x p o l y p he n o l i c f l av o n o i d
mat e r i al p r e s e n t i n t e a i n f u s i o n s i n v i e w o f t he f ac t t hat t he c he mi c al u n i t s o f
w hi c h t hi s mat e r i al i s mai n l y c o mp o s e d ar e c l o s e l y r e l at e d i n s t r u c t u r e t o t he p o l y -
p he n o l i c u n i t s p r e s e n t i n s e v e r al c ar c i n o g e n i c v e g e t abl e t an n i n e xt r ac t s ( K i r by ,
1 9 6 0 ) . The an al y s i s o f t hi s mat e r i al , w hi c h t o o k ac c o u n t bo t h o f t he maj o r p o l y -
p he n o l i c c o mp o n e n t s i n a q u an t i t at i v e w ay an d o f t he man y mi n o r c o mp o n e n t s
s e mi - q u an t i t at i v e l y , s ho w e d t hat t he p r e s e n c e o f c al c i u m i n w at e r u s e d t o p r e p ar e
t e a i n f u s i o n s has n o mar k e d e f f e c t o n t he e xt r ac t i o n o f an y o f t he p o l y p he n o l i c
1 1
1 2 W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
s u bs t an c e s s t u d i e d . The r e i s t he r e f o r e n o e v i d e n c e f r o m t he p r e s e n t s t u d y o f an y
i n t e r e s t i n g l i n k be t w e e n t he s u p p o s e d w at e r f ac t o r i n t he ae t i o l o g y o f c e r t ai n f o r ms
o f mal i g n an t an d c ar d i o v as c u l ar d i s e as e , o n t he o n e han d , an d t he p o l y p he n o l i c
s u bs t an c e s p r e s e n t i n t e a i n f u s i o n s , o n t he o t he r . It may be n o t e d f r o m Tabl e 1
t hat t he s e p o l y p he n o l i c f l av o n o i d s u bs t an c e s c o n s t i t u t e ap p r o xi mat e l y hal f t he
t o t al amo u n t o f s o l i d mat e r i al p r e s e n t i n t he i n f u s i o n s an d t hat t he av e r ag e d ai l y
i n t ak e o f t e a p o l y p he n o l s i n t he Un i t e d K i n g d o m i s abo u t 2 g . The r e c e n t r e p o r t s
( K o r p as s y , 1 9 6 1 ; K ai s e r , 1 9 6 7 ) w hi c h r ai s e t he q u e s t i o n w he t he r t he c o n t i n u al
i n g e s t i o n o f t he s e s u bs t an c e s i n t e a mi g ht p r e s e n t e i t he r a c ar c i n o g e n i c o r c o -
c ar c i n o g e n i c r i s k t o man s ho u l d e n c o u r ag e f u r t he r i n v e s t i g at i o n o f t he i r bi o l o g i c al
ac t i v i t y .
Of t he o t he r t e a c o mp o n e n t s e xami n e d , i t w i l l be n o t e d t hat t he p r e s e n c e o f
c al c i u m i n w at e r r e p r e s s e s t he e xt r ac t i o n o f bo t h o xal i c ac i d an d p e c t i n i c ac i d . It
may al s o be n o t e d t hat a maj o r p ar t o f t he c al c i u m u p t ak e p r o c e s s ( F i g . 1 ) i s
ac c o u n t abl e i n t e r ms o f c al c i u m s al t d e p o s i t i o n o f t he s e t w o s u bs t an c e s i n t he l e af .
It i s p r o babl e t hat t he r e mai n d e r o f t he c al c i u m u p t ak e i s d u e i n p ar t t o c al c i u m
bi n d i n g by i n s o l u bl e l e af c o mp o n e n t s , an d s o me u p t ak e by e xc han g e w i t h l e af
mag n e s i u m may al s o be i n v o l v e d . The i n f o r mat i o n o n t he p e c t i n i c ac i d f r ac t i o n
w as s o u g ht mai n l y t o d e t e r mi n e t he e xt e n t t o w hi c h t hi s f r ac t i o n w as r e s p o n s i bl e
f o r c al c i u m u p t ak e , an d n o t by r e as o n o f an y s p e c i al p har mac o l o g i c al i n t e r e s t . It
i s o f g e n e r al i n t e r e s t t o n o t e t hat , i n f o o d - c an n i n g p r ac t i c e , t he p r o c e s s o f ' f i r mi n g '
o f c e r t ai n t y p e s o f f r u i t an d v e g e t abl e s ( e . g . ap p l e s an d t o mat o e s ) by c al c i u m s al t
t r e at me n t has be e n as c r i be d t o a c al c i u m u p t ak e r e l at e d t o t he d e p o s i t i o n o f l o w -
e s t e r c al c i u m p e c t i n at e s w i t hi n t he v e g e t abl e t i s s u e ( K e r t e s z , 1 9 5 1 ) . Ev i d e n t l y
a s i mi l ar t y p e o f i n t e r ac t i o n o f w at e r c al c i u m w i t h p e c t i n i c ac i d o c c u r s i n t e a d u r i n g
i n f u s i o n p r e p ar at i o n bu t , i n t he c as e o f t e a, d e p o s i t i o n o f c al c i u m as o xal at e f o r ms
a mo r e i mp o r t an t p ar t o f t he u p t ak e p r o c e s s . The r e s u l t s f o u n d o n t he o xal at e
c o n t e n t o f t he i n f u s i o n s me r i t f u r t he r d i s c u s s i o n .
Co mp ar at i v e l y l i t t l e i n f o r mat i o n has be e n p u bl i s he d o n t he o xal at e c o n t e n t o f
t e a i n f u s i o n s . Me as u r e me n t s by B au ( 1 9 2 0 ) i n d i c at e t he o c c u r r e n c e o f abo u t 5 0 0 mg .
o f s o l u bl e o xal at e p e r 1 0 0 g . t e a l e af an d H o o v e r & K ar u n ai r at n am ( 1 9 4 5 ) r e p o r t
t he p r e s e n c e o f abo u t 1 3 5 mg . o xal at e p e r l i t r e i n a t e a i n f u s i o n . The p r e s e n t v al u e s
f o u n d f o r s i x t e a s amp l e s , s ho w i n g a r an g e o f 6 2 - 9 8 mg . o xal at e p e r l i t r e o f
i n f u s i o n , ar e i n br o ad ag r e e me n t w i t h t he s e e ar l i e r r e s u l t s an d c o n f i r m t he g e n e r al
o c c u r r e n c e o f ap p r e c i abl e amo u n t s o f s o l u bl e o xal at e i n bl ac k t e a. It i s al s o c l e ar
f r o m F i g . 2 t hat t he e xt r ac t i o n o f o xal at e d u r i n g t he p r e p ar at i o n o f o r d i n ar y t e a
i n f u s i o n s i s p r o g r e s s i v e l y r e p r e s s e d as t he c o n c e n t r at i o n o f c al c i u m i n t he e xt r ac t i n g
w at e r i s i n c r e as e d . It s ho u l d be n o t e d t hat i n d i s t i l l e d w at e r i n f u s i o n s t he o xal at e
c o n t e n t i s i n l ar g e e xc e s s o f t he c al c i u m c o n t e n t . In i n f u s i o n s p r e p ar e d w i t h w at e r
c o n t ai n i n g mo r e t han abo u t 5 0 mg . c al c i u m p e r l i t r e , o r i n i n f u s i o n s w hi c h hav e
be e n ad mi xe d w i t h mi l k i n t he o r d i n ar y w ay , t he c al c i u m e xc e e d s t he o xal at e i n
e q u i v al e n t s . It ap p e ar s , ho w e v e r , t hat t he o xal at e i s n o t r e ad i l y p r e c i p i t at e d f r o m
t he i n f u s i o n u n d e r o r d i n ar y c o n d i t i o n s an d r e mai n s w i t hi n t he bo d y o f t he l i q u i d
af t e r mi l k ad d i t i o n f o r p e r i o d s o f at l e as t 1 0 mi n . The v al u e s f o u n d f o r o xal at e i n
t he t e a i n f u s i o n s t he r e f o r e p r o v i d e a r o u g h g u i d e t o o xal at e i n t ak e f r o m t e a. The
Te a i n f u s i o n s an d w at e r har d n e s s 8
r e s u l t s i n d i c at e t hat t he av e r ag e d ai l y i n t ak e o f o xal at e f r o m t e a i n d i f f e r e n t p ar t s
o f t he Un i t e d K i n g d o m v ar i e s i n v e r s e l y w i t h t he d e g r e e o f har d n e s s o f t he l o c al
w at e r s u p p l i e s , an d amo u n t s t o s o me 6 0 mg . i n s o f t w at e r ar e as an d t o hal f t hat
v al u e i n ar e as s u p p l i e d w i t h w at e r c o n t ai n i n g mo r e t han abo u t 1 0 0 mg . c al c i u m
p e r l i t r e .
Mo s t o f t he p u bl i s he d i n f o r mat i o n o n t he o xal at e c o n t e n t o f f o o d s r e l at e s t o i t s
o c c u r r e n c e i n v e g e t abl e s an d f r u i t . Us i n g t he o xal at e d at a o f K o hman ( 1 9 3 9 ) t o -
g e t he r w i t h i n f o r mat i o n o n f o o d c o n s u mp t i o n i n t he Un i t e d K i n g d o m f o r t he p e r i o d
1 9 5 6 - 6 5 ( Nat i o n al F o o d Su r v e y Co mmi t t e e , 1 9 6 7 ) w e e s t i mat e t hat t he av e r ag e
d ai l y i n t ak e o f o xal at e f r o m v e g e t abl e s an d f r u i t amo u n t s t o s o me 6 5 mg . , o f w hi c h
abo u t o n e - t hi r d i s c o n t r i bu t e d by p o t at o e s . The f i g u r e s av ai l abl e f o r o t he r f o o d s
( J e g he r s & Mu r p hy , 1 9 4 5 ) s u g g e s t t hat c e r e al s , me at , mi l k , c o f f e e an d c o c o a, t ak e n
as a w ho l e , mi g ht c o n t r i bu t e a f u r t he r 5 0 mg . o f o xal at e t o t he av e r ag e d ai l y i n t ak e .
The s e r o u g h e s t i mat e s i n d i c at e t hat t e a i n f u s i o n s ar e t he r i c he s t s i n g l e s o u r c e o f
o xal at e i n t he Un i t e d K i n g d o m d i e t . What i s n o t c l e ar i s w hi c h o f t he v ar i o u s
o xal at e s o u r c e s ar e t he mo r e i mp o r t an t o n e s i n t e r ms o f t he c o n t r i bu t i o n s t he y
mak e t o t he o xal at e l e v e l i n t he bl o o d , f o r l i t t l e d i r e c t i n f o r mat i o n i s av ai l abl e o n
t he c o mp l e x q u e s t i o n o f t he abs o r p t i o n o f t he s e c o mp ar at i v e l y s mal l amo u n t s o f
o xal at e f r o m d i f f e r e n t f o o d s .
Var i o u s as p e c t s o f t he me t abo l i s m an d o f t he p hy s i o l o g i c al ac t i o n o f o xal at e s
abs o r be d f r o m f o o d ar e al s o p r o bl e mat i c . Oxal ae mi a an d o xal u r i a hav e f r e q u e n t l y
be e n r e p o r t e d as f e at u r e s o f a w i d e v ar i e t y o f d i s e as e s , w i t h l o w - o xal at e d i e t s
r e c o mme n d e d as t he r ap y , al t ho u g h, e xc e p t i n t he f o r mat i o n o f o xal at e c al c u l i an d
t he o c c u r r e n c e o f s e v e r e r e n al c o l i c , t he r e l at i o n be t w e e n t he d i s e as e s t at e s an d t he
t i s s u e o xal at e c o n c e n t r at i o n s has g e n e r al l y be e n i n d e f i n i t e ( J e g he r s & Mu r p hy ,
1 9 4 5 ; No r d i n & H o d g k i n s o n , 1 9 6 7 ) . Ag ai n s t t hi s bac k g r o u n d , i t s e e ms ap p r o p r i at e
t o r e c o mme n d t hat , i n f u r t he r r e s e ar c h o n t he s i g n i f i c an c e o f d i e t ar y o xal at e s ,
at t e n t i o n s ho u l d be g i v e n t o t he o xal at e c o n t r i bu t i o n f r o m t e a i n f u s i o n s an d al s o
t o t he i n f l u e n c e o f c al c i u m f r o m d r i n k i n g - w at e r o n bo t h t he i n t ak e an d t he ab-
s o r p t i o n o f o xal at e s f r o m f o o d s . An e xami n at i o n o f t he p o s s i bi l i t y t hat t he r e may
be s o me c o n n e xi o n be t w e e n t he ' w at e r f ac t o r ' i n c ar d i o v as c u l ar d i s e as e an d t he
abs o r p t i o n o f o xal at e s f r o m f o o d s ap p e ar s t o be a v al i d l i n e f o r f u r t he r i n q u i r y .
We w i s h t o t han k t he B r i t i s h No n - F e r r o u s Me t al s Re s e ar c h As s o c i at i o n , Me s s r s
J o hn s o n , Mat t he y an d Co . Lt d . , G e o r g e J o hn s o n an d Co . ( B i r mi n g ham) Lt d . , an d
t he Ti n Re s e ar c h In s t i t u t e f o r p r o v i d i n g bo t h s amp l e s an d i n f o r mat i o n o n t he
c o mp o s i t i o n o f v ar i o u s me t al s an d al l o y s .
We al s o w i s h t o t han k P r o f e s s o r W. V. May n e o r d , C. B . E. , F . R. S. , w ho i n i t i at e d
t he s t u d y , f o r hi s g u i d an c e t hr o u g ho u t t he w o r k .
1 3
1 4 W. ANDERSON, J . G . H OLLINS AND P AMELA S. B OND
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