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Photosensitivity diseases inNew ZealandE. P. White aa Animal Research Station , Department ofAgriculture , HamiltonPublished online: 14 Feb 2012.

To cite this article: E. P. White (1958) Photosensitivity diseases in NewZealand, New Zealand Journal of Agricultural Research, 1:4, 433-446, DOI:10.1080/00288233.1958.10431528

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NEW ZEALANDof

AGRICULTURAL

JOURNAL

RESEARCH

Department of Scientific and Industrial Research, WellingtonEditor: M. A. Black.

VOL. 1. AUGUST 1958 Number 4

PHOTOSENSITIVITY DISEASES IN NEW ZEALANDXII. CONCENTRATION OF THE FACIAL ECZEMA POISON

By E. P. WHITE, Ruakura Animal Research Station, Department ofAgriculture, Hamilton

(Received for publication, 23 May 1958)

SummaryThis paper describes procedures for the preparation of concentrates

of the facial eczema hepatotoxin from toxic dried grass. Fractions havebeen made repeatedly containing 1/3000 of the weight of the originaldried grass with very little loss of poison, and fractions containing1/20,000 and up to 1/100,000 of the weight with a slight loss of thepoison.

Other information from fractionation studies is given, providingalternative techniques, and giving information on the chemical andphysical properties of the poison.

Evidence is presented that the concentration of the poison inrelatively toxic dried grass is well below the order of 1 part in 100,000.

INTRODUCTION

The presence in New Zealand pastures growing under autumnflush conditions of a poison which causes the disease facial eczema insheep and cattle has been inferred from the nature and occurrence ofthe disease (Filmer 1951; Clare 1952, 1955). Liver damagecharacteristic of facial eczema has in fact been produced in lambs andguinea pigs fed dried grass obtained from pasture that produced liverdamage in lambs grazed upon it (Simpson et at. 1957).

Earlier attempts to isolate a poisonous principle have been delayedbecause of the difficulty of collecting and preserving toxic grass, and bythe lack of a suitable small scale test animal for measuring toxicity.Clare (pers. comm.) in 1949 produced in a lamb slight liver damage,which could not be attributed specifically to the facial eczema poison,by dosing the alcoholic extract of 150 lb of toxic grass over 15 days.

In 1952, following the demonstration that the guinea pig could beused to test for the poison by Evans et at. (1957), work on isolation ofthe active principle was commenced using grass collected the year before.

N.Z. J. agric. Res. 1; 433-446.

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The work of Perrin (1957) on the susceptibility of the guinea pig tothe poison, done concurrently with the extraction work, has allowedfeeding techniques for extracts to be improved during the course of thework. This paper reports the progress so far in the systematic extractionand fractionation of the poison from pasture, and decribes the proceduresthat have been employed.

During 1952 it was established that ether or acetone extracted thepoison from dried grass (Perrin et al. 1953) thus proving that an organicpoison was involved. A few advances such as the demonstration of theneed for "cold evaporation" of solvents were made prior to March 1955when grass of higher toxicity was obtained. Most of the progress reportedhas been made since that date.

Throughout this work, certain general principles have beenobserved. Firstly, because of the small amount of toxic grass availableeach year, and the uncertainty of further supplies, usually only a fewfractionation experiments have been done at a time. Subsequentpossible steps were often developed in the meantime using non-toxicgrass, and decided upon only when results of previous trials wereavailable. Since the location of the poison in fractions required feedinga guinea pig for at least four weeks, and up to six weeks during theearly stages of the work, this system has delayed progress. Recently,with more toxic grass available, many fractionations have been done ata time.

Secondly, as it was thought that the facial eczema poison was likelyto be easily destroyed, and as this was evident in the early stages of thework, the simplest and least rigorous techniques were used. For example,extracts were at first dispersed directly on grass for feeding, and onlylater were methods of obtaining dried toxic extracts developed.

Thirdly, since until recently, grass could not be spared to obtaininformation on the stability of concentrates in solution or in the driedstate, each fractionation experiment has started from the grass. Allprocesses were carried out without delay, with a minimum of storageof solutions or dried extracts.

Techniques have been developed on the basis of splitting theweight of a fraction into smaller well-defined fractions, and only thosein which the poison was repeatedly recoverable without loss in a smallfraction, with no more than traces in the other fractions, were adopted.

EXPERIMENTAL

Grass Samples Used) and Feeding of Extracts

The nine grass samples used in this work were collected in differentyears from several localities, and were from ryegrass-dominant pastures(including in one case short-rotation ryegrass) and have contained smallamounts of white clover and other species.

At first the grass was stored in sacks in a dry place, but the poisondisappeared after eighteen months. Subsequently all toxic sampleshave been coarsely ground in a Wiley mill, and packed into large tins

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which were stored at 0 to -lODe. Under such conditions no loss oftoxicity has been observed within eighteen months.

The amount of grass used in each extraction has been between oneand a half and two times the quantity required to give severe liverdamage when grass was fed direct. Because the samples differed con­siderably in toxicity, and guinea pigs of progressively lower weight wereused during the course of the work, the duration of feeding and weightof grass used has varied considerably. When only low toxicity sampleswere available (1953, 1954) extracts of 2700 g were fed to 160-g guineapigs for six weeks with a few positive results. For the more toxic samples(Claudelands Showgrounds 1955, 1956; Gisborne 1956, 1957) extractsof 550 or 900 g werefed to guinea pigs of 80-100 g initial weight forthree weeks, followed by one week on non-toxic grass. Such extractsconsistently gave severe damage.

Extracts were fed on ground grass in the manner described byPerrin (1957). Extracts in all solvents used except aqueous methanols,can be put on ground non-toxic grass in a large dish, and the solventremoved in an air-blast, with ice-cooling in some cases, without losingtoxicity. Dried concentrates were generally dissolved in ether fordispersal. The weight of grass used was adjusted for the scale of feedingused, and in all later work was 168 g. This gave 21 lots of eightgrammes of preparation, each of which was put in a carton and mixedwith four grammes of milk powder. Usually all the preparation wasconsumed, and an adequate weight gain resulted. Occasionally guineapigs did not survive the four weeks, but no deleterious effects or liverdamage due to the solvents used were observed.

Originally preparations for feeding were made in several batches,but experience showed that preparations were stable at least for someweeks, so the full requirements for each guinea pig were prepared fromone batch of grass, and as a precaution cartons not needed for aparticular week's feeding were kept at -10°c.

Liver damage was assessed as, described by Perrin (1957). In thiswork, visual examination and weight of livers have been the main guides,and these have been adequate. Sections have been kept for histologicalexamination and in a few cases minor damage not detected visually hasbeen shown histologically. Liver damage due to Bacillus bronchosepticainfection is easily confused visually with the predominantly necrotic typeof facial eczema damage sometimes encountered. Visual examinationof lungs, and histology, are important in this case.

Preparation of aX 70,000 Concentrate df the Poison

Details of the concentration procedure are given for 900 g ofgrass, an amount of highly toxic grass suitable for the feeding ofconcentrates to a single guinea pig, on the feeding scale now used. Forother samples, the volumes of solvents, weight of active carbon, etc. arescaled in proportion but the size of the alumina column has been keptconstant for 500-900 g of grass. Larger quantities of grass, have beenprocessed on this basis arid the final concentrates mixed. Equal1y toxicconcentrates have also been prepared to the 3000 level from 3' kg of

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similar grass, using a large metal extractor of the same type as thatdescribed in (1), and processing in one batch apart from step (6) whichwas done in three batches.

1. Nine hundred grammes of grass is ground finely immediately beforeit is to be extracted, and put in extractors of the type describedby Letham et at. (1957), each extractor holding at the most 600 gof grass. The grass is extracted for 8 hours one day and 4 hours thenext day, with ether de-peroxided with acidified ferrous sulphateand distilled.

2. The ether extract is put in a large evaporation dish and ether blownoff in an air duct when the temperature falls to below OOc. At thelater stage (3 hours) the residue is stirred at times, and the dryresidue is stored overnight at below ODe, protected by a polythenebag.

3. The black tarry residue is rubbed up with 230 ml of absolutemethanol for 15 min. The solution is poured off and the extractionrepeated with 3 more portions each of 120 ml of methanol.Addition of a wad of cotton wool helps to break up the insolublepart which is hard and waxy at the end. On a smaller scale itis easier to add a little filter-aid to the ether extract, and to removethe methanol-insoluble part by filtration or centrifugation; this doesnot affect the toxicity.

4. The methanol solution is filtered if necessary and to it is added 27 gof "Darco G-60" activated carbon, and the suspension is shaken atintervals for two hours. It is then filtered, the carbon on the paperwashed with up to a further 75 ml of methanol and some solutionsqueezed out gently.

5. The solution is made up to 600 ml and placed in a separating funnelto which is added 450 ml of b.p, 40-70oc petroleum ether and 150 mlof water. After shaking, the petroleum ether layer is discarded andthe methanol-water phase extracted with four more lots each of400 ml of petroleum ether. Equally good results are obtained usinga large liquid-liquid extractor.

6. The methanol phase is usually stood overnight below ODe, butremains unaltered in toxicity for at least a week. It is put in aone-litre flask of a rotary evaporator under the high vacuumprovided by a Stokes Microvac Pump, with water circulating inthe separator. The flask is rotated in a shallow dish of water andis allowed to "ice-up". After about 5 hours the methanol hasevaporated and the contents of the flasks are transferred to aseparating funnel with ether and the aqueous part extracted withthree more small volumes of ether. Addition of a little common saltwill break emulsions. The ether phase is put in a small dish andevaporated in a powerful air-blast until only a little water remains.A dry residue is obtained by standing for 12-18 hours in a vacuumdesiccator over concentrated sulphuric acid at below 5 mm pressure.It is advisable to stir the dried residue and evacuate for a shorttime to remove occluded water. The residue is often transferred

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before it is quite dry to a crystallising dish using acetone, the acetoneremoved by the air-blast (hair dryer) and the contents dried inthe desiccator, to constant weight.

7. An alumina column 10 in. long and 1 in. diameter is wet-packedwith B.D.H. "Aluminium Oxide for Chromatographic AdsorptionAnalysis" or with recovered alumina of activity between I and II,using ether dried by calcium chloride and distilled or "Analar"ether. The ether-alumina slurry is put in the tube from a polythenewash bottle and the alumina tapped down. It is then washedwith 200 ml of 2% glacial acetic acid in ether and with 300 ml ofdried ether. The solid from step (6) is dissolved in the minimumvolume of ether (about 15 ml), a little sodium sulphate added, andput on the alumina, and elution with ether continued until' 200 mlof eluate has been collected. This is evaporated in a small dishusing the air-blast (hair dryer and fan), and left overnight in avacuum desiccator as used in step (6) until dry. The residue isan orange gum containing yellow pigments, and usually a trace ofporphyrin, or blue pigment derived from the carotenoids. Con­centrates at this stage can be kept without loss of toxicity for upto 3 months in the dark in vacuo, or in the dark at -lo°c, butlose some toxicity when kept in the light in vacuo, and becomenon-toxic on standing in the air and light.

8. The residue is dissolved in a minimum volume of benzene andput on a 6 X 0.5 in. column of activity V alumina, wet-packedwith benzene. Activity V alumina is prepared by placing aluminaof activity I-II in a vacuum desiccator with 13% of its weight ofwater in a dish and turning the alumina at intervals until the waterdisappears. Elution is continued with benzene. The eluate isdiscarded until the lower part of the leading orange zone has leftthe column. The fraction collected is the top of the leading orangezone, the dark zone at the top of this, and the small gap before afaint yellow zone. The eluate is typically 70-140 ml. The benzeneis evaporated in a beaker in an ice-bath using the air-blast, andthe water removed by vacuum desiccation. The residue is a thickyellow-orange syrup.

9. The residue which must not exceed 55 mg is dissolved in 2.5 mlof "AnalaR" carbon disulphide and put on a 9 X 0.4 in. columnof magnesium carbonate (B.D.H. "For Chromatographic AdsorptionAnalysis") / Celite 545 (1 : 1, each dried at 1400 c ) . The columnis compacted by pressure. Elution is continued with carbondisulphide, and the leading dark zone (6 ml) containing most ofthe pigment and weight is discarded. Addition of ether causesa small yellow zone to move down and when this has emerged(e.g. 15 ml), the ether eluate is cold-evaporated and dried. Theresidue is a slightly yellowish-brown gum, weighing from 9 to 18 mg.

The fractionation procedure is. summarised in the following scheme:

Discussion of Extraction Procedure

The methods adopted have been checked repeatedly on severaltoxic grass samples. Loss of poison to the non-toxic fractions has been

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SCHEME OF FRACTIONATION

Concentrations referred to in this paper are the ratio:

weight grass taken (air-dry weight)-- - --;'eight extract (dried) -

900 g grass

I extracted with ether

IExtract Residue, non-toxic

evaporated

dissolved in methanol

IResidue (concn. 25)

1-.-.Residue, non-toxic

IMethanol phase

evaporated

Residue (concn. 450)

Solution (concn. 50)charcoalized (concn. 100), dilutedwith water, extracted with petroleumether

Petroleum ether phase,non-toxic

Dissolved in ether, put on aluminacolumn deactivated with acetic acid,200 ml ether eluate collected,evaporated.

Residue (concn. 3000)

Put on alumina V column inbenzene, eluted with benzene

Up to and including partof first orange zone,non-toxic

ILater part of orangezone, dark zone, andsmall gap

I evaporated

Residue (concn. 20,000)

ILater zones,non-toxic

Put on magnesium carbonate/celitecolumn in carbon disulphide, elutedwith carbon disulphide, then ether.

Dark zone from carbondisulphide, non-toxic.

Ether eluate

evaported

Residue, toxic (concn. 70,000, 13 mg.)

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remarkably slight, and all such fractions have been fed repeatedly atthe usual level, and at levels corresponding to about three times theamount of grass used, without toxicity being detected in them. Thesubstantially quantitative nature of the recovery of poison at each stephas been checked repeatedly by dispersal of solutions on grass. Commentson the units of the previous section are as follows:

( 1) Batch extractions or percolation with cold or warm ether has shownthat even large volumes of this solvent extract less than half ofthe poison. Continuous methods are necessary. Continuousextraction with acetone is effective in removing the poison, and inconcentrates"the poison is readily soluble in organic solvents. Theextracted grass-.has been fed for a long time, fortified with etherextract of non-toxic grass without showing toxicity.

The use of copper or stainless steel gauze, or aluminium ortin foil packing in the extractors did not alter toxicity of extracts,and a tinned copper extractor has been used successfully.

(2) For the later stages of concentration of ether or other solutions, theair-blast technique, keeping the solution at about OOc, leads tono loss of poison, ordinary techniques destroying it. Thedriedextract can be taken up in ether and a non-toxic "wax" fraction,constituting about a quarter of the weight, centrifuged off.

(3) The methanol-insoluble part contains waxes and much pigment.

("1) The amount of this particularly active carbon added is adequateto remove the bulk of the pigment and to leave a solution com­paratively light in colour and containing some carotenoids andporphyrin. Complete removal of colour by carbon has never lefta toxic solution, nor has the poison been extractable from thecarbon or the carbon found toxic when fed. Evidence on furtheruse of carbon as columns for chromatography is presented laterand supports the undesirability of more complete removal of colourat this stage.

(5) When 95% methanol was USE;d, up to a third of the poison wasfound in the petroleum ether phase (using 3 lots of 2/3 volumes ofpetroleum ether). Carotene, some porphyrins, and glyceridesare removed by the petroleum ether. Dried extracts at variousstages taken up in various volumes and boiling points of petroleumethers, showed that with practical volumes the poison was splitbetween soluble and insoluble fractions, and the best concentratesare characterised by low solubility in this solvent. The methanolphase contains much yellow pigment, with usually a little porphyrin.

(6) Equally good recovery of poison resulted from rotary evaporationto dryness, a slow process, or rotary evaporation with the bathheld at 16°c. A more tedious method is to use the air blast withice-cooling of the evaporating dish, until the temperature cannotbe kept below 6°c, followed by vacuum desiccation taking about36 hours. Satisfactory extraction can be made from 80% methanolusing benzene, or from 60% methanol with chloroform, followedby cold-evaporation of these solvents.

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In one case, cold-evaporation was taken to a stage whereapparently only water remained. The bulk of the poison was inthe gum which separated, but some was found in the aqueous part.This may indicate a limited solubility of the poison in water.

(7) In the first trials of chromatography on alumina of activity I-II(Brockmann scale-Brockmann and Schodder 1941), a considerableportion of the poison was eluted by ether/acetone mixtures (con­centrations 2000, or up to 4000 in finer fractions), and noneobtained by subsequent methanol elution. Subsequent experienceshowed that ether, acetone, or ether/acetone eluates and fractionsof these were generally non-poisonous.

A large number of modifications were tried, designed tofind the factor responsible for the loss of toxicity. The modifica­tions included running the column under reducing conditions(stannous chloride alumina (Kofler 1947), hydrogen sulphide,pyrogallol), at lower or higher temperatures, (0 to + 2°0) 27°0),in darkness, with various solvents (dry ether, wet ether,ether/methanol, petroleum ether/acetone), the use of shortercolumns (5 in. long), and of neutral alumina of activity I (Ledererand Lederer 1954; Stoll 1947) . No improvement was obtained withthese changes. A small concentration (X 3) was achieved witha short column and methanol as eluant.

Use of de-activated alumina. A column was washed withacetic acid in ether thus reducing the activity to about V andalso neutralising in situ. Alumina was also de-activated toactivities II, III, IV, and V on the Brockmann scale, by absorptionof known weights of moisture from water in a dish, the aluminabeing spread out in a vacuum desiccator.

The acetic acid washing experiment gave a severely damagedliver and a concentration, using ether elution, of 3500 times.Activities II and III still gave no toxicity in ether-acetone eluates,but activity IV gave a moderate liver damage, and activity V asevere liver damage. Thus it would appear that alumina asformerly used was of too high an adsorptive activity and thatprovided alumina of activity about V is used destruction of poisondoes not take place. There are many examples in the literatureof changes in substances brought about by high-activity alumina.

Concentrations achieved using ether containing 25% acetoneelution were, with activity V, 2660; with activity IV, 3400; withactivity III, 3400; and with activity II-I, 4000; compared withthe average figure for the acetic-washed alumina using ether aloneof 3000. The important point is that lowered-activity aluminastill allows a good concentration.

The acetic acid washing technique has been adopted, andthe reliability of the procedure shown conclusively by the largenumber of severeIy damaged livers found in fractionation experi­ments including this stage. Unfortunately, with de-activatedalumina and ether elution, resolution into defined zones is notclear, and there is no potentiality of collecting finer fractions. The

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(8)

(9)

poison is eluted near the front of the yellow area on the column,and 200 ml of eluate leaves a margin of safety.

The poison has not been detected in the colourless and later yelloweluate before the first major orange zone, or in the lower part ofthe orange zone, but a trace was detected in the small gap justabove the dark zone. There was none in the small yellow zoneimmediately following the gap. It is not normally practicableto cut the zones collected more finely.

Many modifications tried, including larger columns, use ofalumina containing 16% or 20% water, use of aluminade-activated by acetic acid in benzene sufficiently to stop destructionof the poison, or ~ use of petroleum ether/benzene mixtures gavegood recovery of poison in certain zones, but resolution into zoneswas not so clear, and lower weight concentrations were obtained.

There is a persistent small loss of poison which is not avoidedby running the whole operation in the dark. When the routinestep was repeated to give finer fractions, no poison was recoverable.

When the weight of residue from the previous, stage on occasionsexceeded 55 mg, the poison usually appeared entirely in the darkzone, and close to it, giving little concentration. Many variationsof conditions showed the details given to be the best, and a likelyprocedure for the larger weights is to use two similar columnns eachloaded with half the extract. The poison appears in the laterpart of the eluate near the small yellow zone which forms nearthe top of the column. Finer cutting of the eluate did not alwaysconfine the poison to a defined area of the column.

OTHER INFORMATION ABOUT THE POISON

Effect of Acid and Alkali

Ether solutions of concentrates were used to secure informationon basic or acidic properties of the poison. The toxicity remained inthe ether phase after shaking with several small volumes of approximately1% aqueous HC1, or with 2.5% Na2C03, or 1%NaHCOa in water.The aqueous part was used as a mixing water for non-toxic grass infeeding to guinea pigs. Extractions with 20-60% hydrochloric acid toextract porphyrins gave no recoverable toxicity. The concentrate at20,000 times when treated with a minimum quantity of 85% sulphuricacid to remove carotenoids (Parker and McFarlane 1940) left no toxicityin the benzene.

Ether solutions were also extracted rapidly with 5% aqueoussodium hydroxide or shaken with aqueous sodium hydroxide for 24 hoursto give a "cold" saponification, in one experiment with pyrogallol presentto prevent autoxidation (as used for tocopherols-Tosic and Moore1945). Normal saponification was also done. None of the variousfractions recovered from these experiments was toxic.

Use of Carbon and Carbon-column Chromatography

The addition of carbon to give complete decolourisation of purifiedconcentrates in methanol has not left a toxic solution. Concentrates

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at various stages were put on Darco-Celite columns in methanol, andeluted with methanol, benzene, and quaternary detergents, and in somecases a reducing atmosphere was provided by hydrogen sulphide. Lowlevels of poison have at times been recovered in methanol or benzeneeluates, and the residual carbon has been fed direct, or Soxhlet extractsmade with many solvents without recovering the poison.

Use of Other Chromatographic Columns

Using ether as solvent and eluant after the 450 stage, otherchromatographic media were used, e.g. magnesia/Celite, calciumcarbonate, silica gel, magnesium silicate, giving good recovery of poisonwith little concentration.

After the 3000 stage other media were tried as an alternative toalumina V, using benzene as solvent, and this with more polar solventsas eluants. Concentrations of about 8000 times were obtained frombenzene/ether fractions using Florex XXX/Celite, Floridiny'Celite,Magnesol/Celite, Florisil, Silene EF /Celite, and silica gel, usually withsome loss of poison and zones were not easily defined. Floridin hadthe best potentiality.

After the 20,000 stage were tried magnesium carbonatey'Celitewith carbon tetrachloride, alumina containing 13-20% water with carbondisulphide /benzene, magnesium oxide, calcium hydroxide/Celite, andsilica gel with carbon disulphide./erher. No poison was obtained intotal eluates or fractions.

Paper Chromatograms

Using the carbon tetrachloride/methanol/water system describedin the next section, and the whole of a 20,000 concentrate put on a largeWhatman No. 3 disc, as a two-inch-diameter ring using an air blast,the poison was recovered from the main yellow ,zone near the solventfront, with little concentration. Cellulose powder columns done invarious ways using this solvent system have at times shown poor recoveryof poison, usually none. It has not been possible to recover poison fromthe ethyl oleate system.

Counter-current Distributions

Counter-current distributions usmg 30 small separating funnelsas tubes have been tried after the 3000 stage. The solvents were aqueousmethanol and various non-polar solvents, and were:

(1) Carbon tetrachloride/methanol/water, 62: 35: 3.15 as used by Coleet al. (1953) for lipids,

(2) Petroleum ether b.p, 40-60oc / benzene / methanol/water,65:35:90: 10.

(3) Chloroform/methanol/water, 50: 40: 15.5 (all ratios are volumemeasurements) .

Each distribution used 15 ml of each phase and double the usualfeeding level of the 3000 concentrate. Each fraction was cold-evaporatedand weighed, and beakers grouped to give 6 fractions for feeding. Except

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for the carbon tetrachloride system which showed more spread, andmay have given several peaks if many more tubes had been used, thebulk of the weight was well defined in one peak. Poison was recoveredin each case but was on the peak of weight, so that little concentrationwas achieved.

A lO-tube counter-current using carbon disulphideymethanolywater50: 45 :5 was done after the 20,000 stage. The poison was in the firsttubes containing most of the weight, but most of the colour moved withthe carbon disulphide phase. It is concluded from these distributions,and from paper work, that the bulk of the components and the poisonhave very similar partition coefficients in the above solvent systems, andthat many tubes would be needed to give a separation.

COMPARISON OF CORRESPONDING FRACTIONS FROM TO'XIC AND NON-TOXIC

GRASS

Comparisons have been made between fractions known to bepoisonous, and similar fractions from non-toxic grass, in the hope offinding obvious differences that might be associated with toxicity.

Perrin (pers. comm.) compared weights, and absorption spectra inthe visible and ultra-violet, of concentrates at the stages up to 3000times, also fractions from high- and low-activity alumina using ether,and ether/acetone, without finding pronounced differences. Similarwork on fractions obtained during development of concentrationtechniques led to the same conclusions. This work showed the presenceof substance m.p, 260°c, in toxic samples but not in non-toxic samples,and this substance, which is not the poison, has provided the basis forthe "beaker test". (See Part XIII) .

The ultra-violet spectra of toxic and non-toxic concentrates atthe later stages, of fractions of each, of the 70,000 concentrate, andof fractions beyond this, have also failed to produce any differenceconsistently associated with toxicity. The best concentrates show verylittle absorption in the ultra-violet.

At the "3000" stage the concentrations achieved with routine runs,using 7 grass samples of different toxicities and 3 non-toxic sampleswere in the range 2000 to 6500 (total 57 runs), and with the samesamples at "20,000", 16,000 to 45,000 (22 runs). The next step alsoproduced concentrations in the range 50,000 to 120,000 from toxic ornon-toxic grasses. The higher concentrations were not always fromnon-toxic samples. No weight differences were found in fractions beyond70,000.

Paper chromatograms using the disc technique (White 1957)have been investigated to compare toxic and non-toxic fractions. Themain guide has been ability to resolve the yellow pigments or substancesfluorescent under ultra-violet light. With many conventional systemsor with papers impregnated with Vaseline, paraffin oil, or silicones, orwith acetylated papers, the yellow pigments have remained togetherat the origin or at the solvent front, or have tailed badly without showingworthwhile separation.

Olive-oil impregnated paper with aqueous alcohols as developingsolvent has been used to resolve carotenoids (Nunez 1954). Papers

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impregnated with vegetable oils, or preferably ethyl oleate, or glycolstearate, "Quilon", rubber solution, or rubber latex in conjunction with80% or 90% methanol have shown the presence of several yellow orfluorescent components, but none have revealed distinctive differencesbetween extracts of toxic or non-toxic grasses at the 3000 or 20,000stages.

The solvent system carbon tetrachloride/methanol/water 50: 41: 9,the bottom phase being put in the desiccator and the top phase asdeveloping solvent, gave good resolution .of yellow pigments onunimpregnated paper. At the 20,000 stage this system gave 3 mainyellow zones, and 2 minor incompletely resolved ones. The number,position, and appearance of the zones were the same for toxic and non­toxic preparations. These systems do not give clear resolutions ofpigment zones at the 70,000 stage and beyond.

So far nothing has been detected on papers by staining withreagents, except on the yellow zones. Oxidising agents, particularlyaqueous or acetone permanganate, or chromic acid in acetic acid, showstained areas due to yellow pigments or to high enough concentrationof substances m.p. 1490 or 260°c.

DISCUSSION

The results presented are from about 240 fractionation experimentsusing 700 guinea pigs.

The lesions obtained in guinea pigs by feeding extracts aremacroscopically and histologically identical with those produced byfeeding the dried grass. These are compatible with lesions in lambs fedthe same dried grass and in sheep grazing toxic pasture (Evans et at.1957). The type of liver damage is not known to be produced by anychemical from synthetic or plant sources, fed at dose levels comparablewith those of the facial-eczema poison.

The fractionation procedure to give concentrations, of 50,000to 100,000 times has been checked many times. About half the poisonhas been recovered, the loss occurring in the alumina V step, and nobetter alternative has been found. Concentrates at the 20,000 level havebeen made dozens of times using several toxic grass samples.

Occasional preparations at any stage do not give liver damage,but whether this is due to destruction in manipulation, or to guineapig variation is not known. The evidence from this work and fromthat of Perrin (1957) is that guinea pig variation is not serious at thehigh level of poison being used.

The :Poison is stable in methanol solution for at least some days,and dried concentrates at the 3000 level can be kept for months in thedark in vacuo or at low temperature. The main instability occurs inthe later stages of concentration of solutions, and special techniques weredevised to overcome this. There is also ready destruction of the poisonby high-activity alumina or much active carbon. Considerablemanipulation of solutions after the 3000 stage is possible without anyevidence of instability due to air or light, and at no stage has the

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1958) WHITE-PHOTOSENSITIVITY DISEASES 445

proVlSlon of reducing atmosphere prevented loss of poison. Beyondthe 20,000 stage many seemingly mild techniques lead to complete lossof poison, but some methods using carbon disulphide give a good recovery.

The poison, at least in highly purified concentrates, is readilysoluble in ether, acetone, benzene, methanol, 80% methanol, chloroform,and carbon disulphide, fairly insoluble in petroleum ether and water.

Results of acid and alkaline extractions from organic solvents showthat it is not a base or typical alkaloid, or free organic acid, and isneutral. Concentrated acids destroy it. It is quickly destroyed bydilute sodium hydroxide, and, if an ester or glyceride-like substanceor lactone, it is one 'insoluble in petroleum ether. Tests for esters orlac tones in concentrates-have been negative. Large numbers of typicaltypes of naturally occurring organic compounds are ruled out, forexample it is not a typical protein, peptide, phosphatide, or porphyrin,and is unlikely to contain aromatic nuclei. Traces of yellowish pigment,possibly carotenoid, but not flavonoid, are present in the best concentrates,and no toxic preparation has been colourless.

The properties are consistent with those of a neutral organiccompound, with little if any colour, of medium molecular weight, anddevoid of characteristic absorption in the ultra-violet. It is likely tobe almost saturated, and to contain alcoholic or similar uncharacteristicoxygen functions to account for low solubility in petroleum ether.

Corresponding fractions from toxic and non-toxic grasses showa remarkable similarity in weight, absorption spectra, the nature ofpigments, and other properties, even at the 70,000 level and beyond. Ifthe poison has characteristic chemical and physical properties it mustbe present in the 70,000 concentrate in minute concentration. Thereis the possibility that an optical difference could account for toxicity.

A substance of m.p. 149°c has been isolated from the 3000 stageconcentrates of toxic and non-toxic grasses. A second substance ofm.p. 260°c has been obtained from similar concentrates and is generallyfound in toxic samples only. The isolation and characterisation of thesesubstances will be described in a subsequent paper (Part XIII). Thesesubstances are close to the poison on chromatographic columns but thereis no evidence that the poison is related chemically to them, and datafrom feeding experiments, particularly the fact that they have beenlargely removed from the more refined concentrates, indicates that theyare not the poison. Substances capable of drastic hydrolysis to aminoacids, as is substance m.p. 260°c, are present in both toxic and non-toxicconcentrates at the 70,000 level, but in very low concentration. At leastfive amino acids could be found in such hydrolysates, with no obviousdifference associated with toxicity. If the poison is allied to the substancem.p. 260°c, it must be in the best concentrates in very low concentration.

Work on fractionation after the 70,000 stage is continuing along themajor lines of finer adsorption chromatography, disc-paper chromato­graphy, cellulose powder column chromatography and counter-currentdistribution. So far a further routine concentration step has not beenmade, but on two occasions recoveries of about half the poison of the

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original &"rass have been obtained at concentrations of 300,000 and450,000 tunes. This means that from 900 g of highly toxic grass halfthe poison has been obtained in two to three milligrammes of faintlyyellow-coloured gum.

The poison is present in very low concentration (on recent evidencebelow 5 p.p.m.) in dried pasture that produces severe liver damage inguinea pigs. The concentration in grasses in which lower levels oftoxicity are detected by high-level guinea pig feeding must be much less.It must be a substance of very potent biological activity, and of a highlyspecific nature.

ACKNOWLEDGEMENTS

Throughout this work considerable discussion and helpfulsuggestions have come from Mr. N. T. Clare and Dr. D. D. Perrin.Certain observations quoted in the paper were made by Dr. Perrin,particularly some absorption spectra studies, and the recognition ofthe presence of substance m.p. 260°c on alumina columns.

Mrs. Jean Sandos carried out experiments on extracting the poisonwith ether by batch and percolation methods.

I have to thank Mr. N. M. Lauriston for preparing and recoveringthe large volumes of solvents used, and various people for routine feedingand care of guinea pigs, particularly Miss G. J. Falconer and Miss G.Hughes.

REFERENCES

BROCKMANN, H.; SCHODDER, H. 1941: BeT. dtsch. chem. Ces. 74B: 73.CLARE, N. T. 1952: "Photosensitization in Diseases of Domestic Animals"

p. 24. Commonw. Agric. Bur., Farnham Royal.----1955: "Advances in Veterinary Science", Vol. 2 pp.

182-211. Academic Press, New York.COLE, P. G.; LATHE, G. H.; RUTHVEN, C. R. l 1953: Biochem. J.

54: 449.EVANS, J. V.; McFARLANE, D.; REID, C. S. W.; PERRIN, D. D. 1957:

N.Z. ]. Sci. Tech. A38: 491, 680.FILMER, l F. 1951: Proc. Specialist Con]. Agric., Adelaide, p. 320.

H. M. S.D., London.KOFLER, M. 1947: Helv. chim. Acta 30: 1053.LEDERER, E.; LEDERER, M. 1954: "Chromatography" p. 21. Elsivier,

Amsterdam.LETHAM, D. S.; PERRIN, D. D.; RONALDSON, J. W.; WHITE, E. P.

1957: sz. ]. Sci. Tech. B38: 695-6.NUNEZ, G. 1954: Bull. Soc. Chim, biol., Paris 36: 411.PARKER, W. E.; McFARLANE, W. D. 1940: Canad. ]. Res. B18: 405.PERRIN, D. D. 1957: N.Z. J. Sci. Tech. A38: 669.PERRIN, D. D.; WHITE, E. P.; CLARE, N. T. 1953: Proc. N.Z. Soc.

Anim. Prod. 12: 121.SIMPSON, J. E. V.; SINCLAIR, D. P.;SWAN, J. B.; FILMER, J. F. 1957:

N.Z.]. Sci. Tech. A38: 947.STOLL, M. 1947: Helv. chim. Acta 30: 991.TOSIC, l; MOORE, T. 1945: Biochem.]. 39: 498.WHITE, E. P. 1957: N.Z. J. Sci. Tech. B38: 707-9.

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