The complexing of calcium and magnesium by organic plant constituents : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Chemistry/Biochemistry Department, Massey University

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\

T H

E

^{C 0 M P} L E X I N

G 0

^{F C} A L C I U H

AN D M A G ^{N E S} I ^U M

B y

^ORGANIC

P L

^{A N T C}

⁰

N S T I T U E N T S

LESLIE FRM�CIS 1·iOLLOY

A thesis presented in partial fulfilment of the requirements for the degree of

Doctor of Philosophy

Che�istry/Biochemistry Dcp�rtment Kassey University

February

1971

i ABSTRACT

'l'he definition, occurrence and aetiology of hypomagnesaemic tetany is discussed as an introducti.on to the practical imp­

lications of the present investigation. The current hypotheses, involving an unfavourable pasture chemical composition, account­

ing for the binding of Ca++and l·'lg+-rin the intestlnal tract

o f

ruminants are reviewed. The role of undigested or partly­

digested plant cell wall materials is advanced as another such hypothesis, and the present investigation of the cell wall polymers of a t-ypical pasture grass is outlined.

The comprehensive analysis of the grass Yorh:shire fog

(

Holcus lanatus

)

involves, initially, the extraction and

purification of pectic substances, lignin, hemicelluloses and cellulose. The non-volatile organic acid content of the grass is also determined,

Analytical methods are developed and evaluated in order to assess the homogeneity of these isolated cell wall fractions,

and their chemical constitution investigated to aid in determining any possible relationship between cation complexing and polymer

( o r monorner)

structure.

The pectic fraction isolated from Yorkshire fog is approx­

imately

90%

polygalacturonic acid while the hemicelluloses are basically arabinoxylans with varying hexose and uronic acid content. Attempts to fractionate the predominant hemicellulose, hemicellulose B, into homogenous arabinoxylans gives inconclusive results. Most

o f

the chemical evidence, however, indicates the presence of three discreet polysaccharides in this fraction - a simple arabinoxylan, an acidic galactoarabinoxylan and a neutral glucan.

Infrared and ultraviolet spectroscopy is utilised to

det ermine the puri ty o f the isolat e d lignin as well

as

t h e presen c e o f t ypic al lignin functional groups. Yorkshir e fo g lignin

has a

mo derate phenolic hydro xyl and -01-:e con t en t

_and,

like most o th er monoco tyledonous lignins , gives yie

l

d

s

o f

I

syringaldehyd e , vanillin and p-hydroxybenzald ehyde on alkaline

nitrob enzene o xi dation. The outstanding featur e o f t he lignin is

i

ts

apprec i able

content o f etheri fi e d hydroxyl groups i n the 4-posi tion o f the aromatic ring and the low yield of

s

yri

ng�

aldehyd e .

T h e water-soluble , non-volatil e organic acids

a

r e

quantitatively

det ermined

by anion-exchange

r esin chromatography and their i dentity confirmed by pap er chromatography . The

major acid is the tri carboxylic acid , trans-aconitic acid, which

is

d e t ermined spectropho tometrically . The normal pl

an

t acids,

citri c and mali c ,

are present in

mo derat e q

u

antiti

e

s while

the

ali c y clic

acids , quinic anc shikimic , are only pr esent in minor amounts.

A limi t ed amount o f

dat a

o

n

t h e seasonal fluctuation o f t h ese organic frac tions

in

Yorkshir e fo g i s present ed .

Elec trolytes and the concep t o f ionic activity are discussed i n the intro duction to th e study o f the

ability

of t h es e

pl ant

fractions to bind

Ca + + an

d

Mg+ +

i

n

an aqueous salt solution o f

c ationi c

c omposition similar t o that o f the int estine o f a

ruminant .

A c ation

exchange method is developed wher eby changes in the

a c

t i v

i

ty o f

ea++

or Mg++o n the introduction o f

a

p

lant

fraction into the salt solution are r e flec ted

in the c a tionic composi tion

o f the equil ibrium r esin . An

investigation

is und ertaken o f

a

l arge number o f calibration solutions varying in �a++J and �-ll+], but

c o nstant

in [Na+], [K -+'] and �·m:J,

the lat t er cations being pr esent in exc ess as 'swamping '

c ations .

\

iii

Regression expressions relating solution cation concentration to the equilibrium resin cation concentrations are derived and used as calibration equations to determine the amounts of bound and ionic ea++ ru1d Mg++ in solutions in equilibrium with the plant fractions.

The pectic substances, lignin and the organic acids are effective in complexing a large proportion of the soluti on Ca

in a non-ionic form but only lignin and the organic acids

display a significant cooplexing of solution Hg ++ ^• Except for

hemicellulose B

(

^branched

)

at a slightly alkaline pH, the hemi-

celluloses and cellulose have little ability to complex either ++ _T ++

Ca or Lg. ^•

The complexing results are discussed in terms of the relationship of polymer structure to observed cation affinity.

Factors involved in cation binding are:

- the charge and degree of hydration of the catic·n itself;

- distributi on and degree of esterification of carboxyl groups i n the polymer;

- monomer conformation;

type of glycosi dic linkage in the polymer;

- the possibility of hydrogen-bonding and non-bonded inter- actions between substituents on the polymers;

- soluti on pH.

The in vivo implications of the results are finally dis- cussed in the context of genera.l r uminant nutrition and alkaline- earth metal absorption discussed in the introduction.

A CK

:� 0 '.'.'J, E

DG ;::E?�T S

I wish ^{to thank} my supervisor, Professor

E.

_L. Richards for his encouragement and assistance in this project; I am

also grateful to Professor G. N. Malcol� and Dr. P. Buckley of the Chernistry/Bioche�istry Department for valuable discussions on aspects of the investigation.

The valuable assistance with the statistical treatment of the co�plexing data, rendered by Mr W. B. Currie, Dairy

Husbandry Department, is gratefully acknowledged.

Thanks are also due to Professor Watkins, Agronomy

Department for supplying tl e Yorkshire fog plots; to Dr. R. L.

Bailey, Applied Bioche�istry Division, D.S.I .R., for standard sugar samples and

a

dvi^ce on analytical methodology; to my fellow research students, l·!ike Tirr:perley, Rex Gallagher

and

Jl.^lan ^Danks for many ^helpful discussions; and to the other staff of the

Chemistry/Biochemistry Department for their assistance in many ways.

To my wife, ^Sonia, who could have spent many more valuable hours at music practise had she not been typing this manuscript.

Finally, I wish to express my sincere gratitUde to the Soil Bureau, Department of Scientific and Industrial Research

for the finance which made this investigation possible.

A B

^STRACT

ACKN01'lLEDG:LENTS

CONTE:HTS

LIST ^OF TABLES

LIST OF F

^IGU

R

^ES PART I

V

COJ'I^'EN^TS

HYPOI-iAGNESAEl-iiC TETANY ^{AND ITS} RELA'I'IOHSHIP ^TO THE C

H

D-:ISTRY OF DILTARY ^CALCIUN ^AND I":AGNESIUH

1 . 1 .

DEFINITIO:J, OCCUFWENCE ^AND AEI'IOLOGY ^OF

HYPOl'-'iAGNESAEt.JIC TETANY

Hypomagn esa emia

1 .

2.

ABSORPTIQ;-1

OF CALCIUH PJ.JD i·1AGNESIUH FRmi

THE D

I

G

E

ST^IV

E

TRACrr OF ^RUVINANTS

The physico-c hesical ac tivity of calcium and magnesium in the small intestine of a ruminant

1 . 3.

UNFAVOURAELE PASTURE ^C

H

_EI,:I^CAL CO!-:POSITI01' HYPOTHESES

1

^•

3. 1

^• Pro t ein/carbohydrate imbalanc e

1 . 3 . 2 .

ltlgher fat ty acids (HFA)

1

^•

3. 3.

Organic aci ds

1

^•

3. 4 .

Inorganic anions

1 . 3

^•

.5.

Histamine

1

^•

3. 6.

Conclusions

1 . �·

OUTLINE OF ^P

R E

^SENT INVE.STIGATIOH

PART

II

THE

COHPREHENSIVE ^{AHALYSIS OF} THE ORGATHC FRACTIC:S OF ^{YORKSHIRE FOG}

2. 1. IHTRODUCTimr

2.

2. ^{D�YING AHD}

STORAGE

^{OF GRASS}

i iv V

ix xi

1 2 4 7

8

9

1 1

12

1 4 1 5 1 5 1 5

1 8

1 8

2.

3.

EXTRACTION AHD PURIFICATIOn PROCEDURE

2.3. 1.

Priliminary extractions

2. 3. 2.

Pectic substances

2.3.3.

Lignin

2.3.4.

Hemicelluloses

2. 3.5.

Cellulose

2.3. 6.

Organic acids

2.

4.

ClU11ICJ'J., ANALYSIS OF CARBOHYDRATE FRACTIONS

2.4.1.

Analytical methods

Hydrolysis of polysaccharides Paper chromatography

Quantitative estimation of sugars (a) Spectrophotometric

(b)

Gas-liquid chromatographic

Comparison of spectrophotometric and gas-liquid.

20

20 20 21 22 24 25 25 25 25 25

26 27

chromatographic methods

31

Uronic acid analysis of polysaccharides

32

General polysaccharide purification and analysis

34 2. 4.2.

Results and discussion

2. 5.

CHEf.:ICAL ANALYSIS OF LIGNH�

2. 5.1.

Analytical methods

Nitrobenzene/alkali oxidation of lignin Chrorr.atography of oxidation products

Spectrophotometric analyses

2.5.2.

Results and discussion Ultraviolet spectroscopy

Infrared spectroscopy

Alkaline nitrobenzene oxidation Conclusions

2.6.

ANALYSIS OF OP.GAXIC ACIDS

2.6.1.

ft�alytical methods

Anion exchanse chromatography Paper. chromatography

2.6. 2.

Results and discussion

34 41

41 41 42 42 43 47 49 52 53

53

54

55

vii

2.

7 . COHSTITUENT

CR£-:ICJI..L ANlt:LYSIS OF

YORKSHIRE

^FOG

2.7. 1.

^Res^ul

t

s

and discussion 60

PART III

THE

COl-:PLEXIlYG

^O

F Cr:/"" AND 1,:,/+

D ^{BY THE}

ORGANIC co::STI'l'UE?ITS OF YORKSHIRE

FOG

3.1.1. Introduction 63

3.1 . 2. Electrolytes and the concept of ionic activity 64

Standard salt solution 68

3. 2.

^I

O N

^EXCHANGE HETHOOOLOGY

7 0

3c2.1. Cation exchange equilibria 70

3.2.2. Resin equilibration with standard salt solution 72 3.2.3. Preparation of resin eluates and cation analyses 75

3.2.4. Resin calibration 7 9

3.2. 5. Equilibration with complexing ligand 79

2•3•

^RESULTS

81

3.3.1. Effect of ligand concentration 81

3.3.2. Resin calibration results 82

3.3.3. Plant fraction complexing 83

3.3.4. Resin calibration with solutions lower in calcium and magnesium concentration

Calculation of c&+

fractions Errors

PART IV DISCUSSION

bound by plant

88

89 89

!±. 1 .

^GENERAL

DISCUSSION

^OF HETHODOLOGY AI-ID

RESULTS 92 Plant fraction complexing

4.2.

RELATIOnSHIP OF POLYHER STRUCTURE TO CATION AFFINITY

95

viii

4. 2 . 1. Pectic substances and polyuronides Nature of the cation-uronide linkage

96 99

Configuration of polyuronides 100

Hydrogen bonding and steric factors involved

in cation binding 10 4

Structure-function relationships in monouronides 107 4. 2. 2. Hemicelluloses and other polysaccharides

4.2. 3. Lignin

4. 2 . 4. Organic acids

4 . 3.

^{POSSIBLE IN VIVO} IHPLICATIONS OF COHPLEXUIG RESULTS

APPENDIX I APPENDIX II REFERENCES

10 8

114 115

117

121

126

129

NUMBER

1

ix

LIST OF TABLES TITLE

R

r amnose h

VALUES OF SUGARS IN TWO CHROHA'l'OGRAPEI C SOL VENTS

2

^{RETENTION Til1ES AND} PEAK/ AREA RATIOS OF

3

4

5

ALDITOL ACEI'ATES FRON POLYSACCHARIDE$

CONPARISON OF ME�HODS OF ANALYSIS OF MONO- SACCHARIDE COtlPOSITION OF HEtHCELLULOSES

CHENICAL ANALYSIS OF POLYSACCHARIDE$ FROH YORKSHIRE FOG

HONOSACCHARIDE ANALYSIS OF .ETHANOL-FRACTION­

ATED LINEAR AND BRANCHED HEMICELLULOSE

B

6

PHD�OLIC HYDROl� CONTENT OF LIGNIN FROM YORKSHIRE FOG AS CALCULATED FROH

6£

^(OH-)

CURVE OF FIG.

5b.

7

CHROHATOGRAPHIC DATA ON AROMATIC ALDEHYDES FROM ALKALINE NITROBENZENE OXIDATION OF LIGNIN

8

NON-VOLATILE ORGANIC ACID CONTENT (%

dry weight)

^{OF FOUR SEASONAL SA}tPLES OF}

YORKSHIRE FOG

9

CONSTITUENT ANALYSIS OF THREE SEASONAL SANPLES OF YORKSHIRE FOG IN % DRY WEIGHT

10

WATER-SOLUBLE CONSTITUENTS OF YORKSHIRE FOG

(5/3/68)

11

^ACTIVITY COEFFICIENTS CALCULATED FOR ELECTROLYTES IN STANDARD SALT SOLUTION

12

^{CATIONIC LOAD OF RESIN IN} EQUILIBRIUM WITH STANDP�D SALT SOLUTION

13

^IONIC CHARGE/RADII RELATIONSHIPS

14

^ATOMIC ABSORPTION OPERATING CONDITIONS AND COEFFICIENTS OF VARIATION AS AN ^{INDEX OF}

· PAGE

26

32

36

46

51

56

61

61 69

7 3 7 3

PRECISION OF DETERl'HNATION OF

5

^CATIONS

7 8

15

REGRESSIONS FOR VARIATION OF RESIN INDI- VIDUAL CATION LOAD

Hn +

^{R WITH SOLUTION}

CALCIUH CONC��TRATION Ca

++

^S

83

NUMBER TITLE

16 INDIVIDUAL AND TOTAL

HEAN

RESIN CATION

17

LOADS (meq/g) AFTER EQUILIBRATION WITH CO!'-:PLEXAN T S

++

tvlEAN

DEVIATION ( in meq/ g) OF RESIN

Ng , Na

+,

K+

AND

NH4

+ LOADS FROH VALUE PRE­

DICTED IN FIG. 13 FOR RESIN Ca ++ LOADS (in meq/g) OBTAINED ON EQUILIBRATION WITH

PAGE

85

PLANT FRACTIONS 86

1 8

HEAl� ^·

R ESIN CATION LOADS (meq/ g) PLUS CORRESPON DING

HEAN

DEVIATIONS (a, descri bed

19

in t able 17 ) FROH Ca++ LOADS PREDICTED FROH FIG

^..

13 AFTER �UILIBRATION WITH COHPLEXANTS

PERCENTAGE OF SALT SOLUTION FRACTIONS (50 mg)

Ca ++ AND

Mg

++ IN STANDARD (20 cm3) BOUND

^BY

PL��T

20 RELATIONSHIP BETWEEN URONIDE CONTENT OF PLANT FRACTI,:)NS AND DEGREE OF Ca

++

AN D

Mg

++ BINDING

87

90

xi

LIST OF FIGURES (fu�D PLATE)

NUMBER AFTER

PAGE

SCHEHATIC DIAGR.A.N OF DIGESTIVE ORGANS OF

A RU!HNANT 6

SCHEME FOR FRACTIO:tJATION OF POLYSACCHARIDES

IN GRASS 20

2 STANDARD ABSORBENCE CURVES FOR SUGARS

3 SEPARATION OF ALDITOL ACETATES FROH HEMI­

CELLULOSE B (linear) ON SE-30

4 ETHANOL FRACTIONATION OF LINEAR AND BRANCHED HEHICELLULOSE B

5 DIRECT (a) AND DIFFERENCE (b) ULTRAVIOLET SPECTRA OF LIGNIN

6 7

INFRARED SPECTRA OF PLANT FRACTIONS

SEPARATION OF AROHATIC ALDEHYDES FRON LIGNIN

& ELUTION SPECTRUM OF NON-VOLATILE ORGANIC

27

38

43 47

51

ACIDS FROH ANION-EXCHANGE RESIN,

_DOWEX

1 56

9 UPTAKE OF CATIONS BY FJtUILIBRATING RESIN AS INDICATED BY EFFLUENT CATION CONCEN-

TRATION ? 2

10 EFFECT OF IONIC RADIUS ON ION EXCHANGE IN A CARBONACEOUS ZIDLITE (Data of Nachod

and Wood) 7 5

1 1 PROGRESSIVE RESI1 ADJUSTHEi T

_TO

NE// £XtUI­

LIBRIUH BY SUCCESSIVE EQUILIBRATIONS WITH A NEW SOLUTI01T

12 CHANGE IN RESIN CATION CONCENTRATION WITH INCREASE IN LIGAND CONCENTRATION

79

81

xii

NUl-iBER

13 VARIATION IN RESIN IHDIVI DUAL CATION LOAD

+ +

WITH CHANGE IN Ca IN CALIBRATION SOLU'l'IOHS

PLATE POLYSACCP.ARI DES AND LI GNIH I SOLATED FROM

AFTER PAGE

82

YORKSHIRE FOG 22