Vol. 50 AUOUBIN AND ASPERULOSIDE 319

Heris8ey, H. & Lebas, C. (1910). J. Pharm. Chim., Pari8,(7),2,490.

Hill, R. & Van Heyningen, R. (1951). Biochem. J. 49, 332.Karrer, P. & Schmid, H. (1946). Helv. chim. Acta, 29, 525.Levine, V. E. & Richman, E. (1933). Biochem. J. 27, 2051.Levine, V. E., Seaman, C. le R. & Shaughnessy, E. J. (1933).

Biochem. J. 27, 2047.

Partridge, S. M. & Westall, R. G. (1948). Biochem. J. 42,238.

Plouvier, V. (1944). C.R. Acad. Sci., Paris, 218, 605.Reichstein, T. (1932). Helv. chim. Acta, 15, 1110.Wettstein, A. & Miescher, K. (1943). Helv. chim. Acta, 26,

788.Zellner, J. (1934). Arch. Pharm., Berl., 272, 601.

The Accumulation and Utilization of Asperuloside in the Rubiaceae

BY A. R. TRIMBiochemical Laboratory, Univer8ity of Cambridge

(Received 14 February 1951)

The significance of glycosides in plant metabolism isobscure, and the present work has been undertakenas a contribution to its elucidation. Large amountsof glycosides are present in many plant cells andalthough these substances vary widely in theirchemical constitution many of them exhibit acommon pattern of distribution in the plant. Theyare accumulated in the vacuoles of young partsof plants during the period of cell expansion, andafterwards may be partly or completely removed asthe part ages.The plants investigated were: Galium aparine

(cleavers, or goose-grass), Asperula odorata (wood-ruff) and Rubia tinctorum (dyer's madder), whichbelong to the tribe Stellatae ofthe family Rubiaceae.Most plants produce glycosides of a number ofaglycone types, and the Stellatae are known toproduce anthocyanins and related glycosides, whichare universal among higher plants, anthraquinoneglycosides, coumarin glycosides and asperuloside,which probably has a substituted furan structure(Trim & Hill, 1952).

Asperuloside was chosen for study because itoccurs in very large quantities in the plants investi-gated, and because its chromogenic propertiesprovide very simple means for its detection andestimation.

EXPERIMENTAL

The methods of growing and collecting the different plantmaterials will be described with the experiments in whichthey were used. The time of collecting was mid-morning.Extracts were made from fresh, weighed material unlessotherwise stated. Dry weights were determined after dryingequivalent batches of the material for several days at 370and then over concentrated H2SO4 and solidNaOH in vacuo.The parts to be extracted were ground with an equal weightof 0-1x-HOl and a little sand. A few drops of CHC13 wereadded and the mush allowed to stand for about 15 min. Theground tissues were then mixed with kieselguhr, filtered ona Buchner funnel and washed with water until the runningswere free from asperuloside. The extract was made up to an

appropriate volume with water, neutralized with CaCO3 andfiltered.

Estimation of a8peruloside. 1 ml. samples of the extractswere mixed with 11 ml. ofreagentA (Trim & Hill, 1952) andheated on a boiling-water bath for 5 min. with occasionalshaking. The characteristic blue derivative of asperulosidedeveloped. Thecooled solutionwascomparedwitha standardin a Duboscq-type visual colorimeter. Beer's Law appliesover a wide range of concentrations as may be seen inTable 1. The effect of the addition of a plant extract is

Table 1. Colorimetric e8timation ofknoum amounts of a8perulo8ide

Recovery, using aAsperuloside 1 mg. standard

(mg.) (%)0-25 1080*50 1000*75 1001.0 100

Table 2. Colorimetric e8timation of known amount8of a8perulo8ide in the presence of an extract ofGalium aparine

Asperulosideadded as Total

Extract 0 1 % solution found Recovered(ml.) (mg.) (mg.) (mg.)0*5 0.0 0*21 0.00*5 0 10 0*31 0.100 5 0*25 0*46 0*250*5 0*50 0-71 0.500.0 0.50 0*50 0.50

shown in Table 2. The colour developed under these condi-.tions has an absorption spectrum with bands at 645 and600 m,. Early in the season, when most parts of the plantswere young, very little extraneous colour was developed.Later, when the bulk of the tissues were older, the plantsproduced sufficient quantities of interfering substances tonecessitate the use of a red ifiter when making the colori-metric measurements. The nature of the interference bysubstances such as phenols, ketoses and heterocyclic Ncompounds, which are likely to occur in plants, has been

A. R. TRIMdescribed (Trim & Hill, 1952). Since relatively large pro.portions of these substances must be added to cause amarked effect, the interference was rarely serious.

Reagent A was also used for the detection of asperulosidein portions of plant tissues. The blue colour develops insections and fragments of plant heated with reagent A on aslide or in a test tube. This procedure was used to detect thelocalization of asperuloside.

Galium aparineThe material was collected at random from one

large plot of plants. Ripe seeds were collected fromthe plot at the end of the season 1946 and used forexperiments during the following winter and spring.

Seeds and seedlings. Batches of 30 newly ripenedseeds were analysed and found to contain 3-5%asperuloside on a dry-weight basis. After storinguntil the following spring the value had fallen to2 5% and the seeds remained viable. Qualitativetests on sections which had been cut from seedssoaked in water for 16 hr. showed that the endo-sperm and the whole of the embryo containedasperuloside. As germination proceeds the endo-sperm shrinks and the amount of asperuloside in itdeclines. The embryo develops at the expense of theendosperm. Asperuloside was found in all parts ofthe developing embryo and was in particularly highconcentrations in the early embryonic hypocotyland cotyledons. These parts gave a purple colourwith reagent A, which suggested the presence ofa large proportion of the interfering substancesmentioned above. The interfering material dis-appeared during the development of the seedling.As the seedlings grew they were dissected into theirparts and each part was tested separately. Thisconfirmed the observation that asperuloside occursin all parts ofthe very young seedling and that thereis a high concentration in the cotyledons. Theglycoside appears to be absent from the growing tipsof the roots.The following measurements show how asperulo-

side accumulates during the period of germination.Two batches of about 500 seeds were sown on wetsand in two shallow dishes covered with sheets ofglass, slightly raised above the tops of the dishes toadmit air. Both dishes were kept in a greenhouse at180 and one of them was covered to exclude light.At intervals two samples of 30 seeds were removedfrom each dish and ground with sand and a fewml. 3 % HCI to extract the asperuloside. This wasestimated, and the values obtained are plotted inFig. 1. The initial fall in the amount of asperulosideper individual takes place while the sproutingradicle has still not emerged through the seed coat.By the time the m-imum has been reachedsprouting is just visible among the seeds which havebeen kept in the dark. These seedlings then accumu-late asperuloside until the amount per individualreaches the level in the ungerminated seed, and if

the seedlings are kept dark this level is maintainedfor many days. By the time the maximum has beenreached the plant has fully emerged. Exposure ofthese seedlings to the light results in an immediatemarked increase of asperuloside, due presumably tophotosynthesis. The sprouting of the seedlingsgrown in the light is retarded by about 8 days andtheaccumulation ofasperuloside is slower, butbythetime the cotyledons appear the amount of asperulo-side has reached the level of the maximum forseedlings grown in the dark. There is then a rapidincrease in the amount of asperuloside due to thesupervention of photosynthesis.

04 . , u u . u

04J

E\O0U 03 t _ ~00

0.

02 I . , , , I

0 2 4 6 8 10 12 14 16 18 20Time (days)

Fig. 1. The production of asperuloside in germinatingseeds of G. aparine: (i) @-, seeds germinated in thedark; (ii) 0-0, seeds germinated in the light.

This experiment was repeated a month later andthe same changes were observed. The amount ofasperuloside in the seedlings germinated in thedark reached 0-34 mg. per plant after 12 days andremained constant for another 10 days. Then it felloff slowly, reaching 0-26 mg. per plant 32 days fromthe initiation of germination. Some of the seedlingsgerminated in the dark were exposed to the light18 days after the beginning of the experiment andimmediately began to produce more asperuloside.After 2 days' exposure there was 0-4 mg. per plantand after 15 days 0 59 mg. per plant.

It is concluded from these experiments thatasperuloside is continuously accumulated by thegerminating embryo in the absence of externalnutrients other than water, the initial rapid fall inthe amount of glycoside being due to the utilizationof the endosperm. This accumulation is terminatedabruptly at a certain level unless photosynthesis ispermitted. The effect of feeding through the rootswas not tested.

320 I952

Vl5ASPERULOSIDEAsperuloside formation in seedlings grown in soil.

Twelve 25 cm. pots of soil were each sown with40 seeds of G. aparine and kept in a greenhouse at180. After a month, when the seedlings were welldeveloped, they were carefully removed from twoof the pots, washed, counted and each plant dividedinto four parts: roots, hypocotyl, cotyledons andshoots. The parts from one pot were weighed and theasperuloside was extracted. The parts from theother pot were used for the determination of thedry weights. Further samples were taken as theseedlings developed. Some of the results are plottedin Fig. 2.

1 4

122

100

_. 08

E-

o 06

04

02

0 4 8 12 16 20Time (days)

hypocotyl were no longer growing, while the shootsand roots were rapidly increasing in weight.

Table 3. Dry weights of parts ofGalium aparine seedlings

Dry weight (mg.)

Day Hypocotyl0 3.312 2*621 1*2

Cotyledons6*65*25-2

Shoots7-4

27-058-0

Roots17-023-058-0

The seasonal variation of the amounts of asperulo-side. Individual shoots from different plants at thesame stage of growth varied considerably in theirasperuloside content. It was found that significantvalues for comparative purposes could be obtainedby analysing batches of six or more shoots picked atrandom from the experimental plot. The values forsix batches of six shoots picked at the same time aretabulated in Table 4.

Table 4. Asperuloside in the shootsof Galium aparine

(Batches of six shoots.)

Batchno.

1

2.3

456

24

Fig. 2. The accumulation of asperuloside in seedlings ofG. aparine grown in soil with normal lighting; (A) leaves,(B) roots, (C) cotyledons, (D) hypocotyl.

As the plant develops there is continuous steadyaccumulation ofasperuloside in the roots and shoots.At the same time, the amount in the cotyledons fallsto a constant low level. While there is a possibility oftransport of glycoside from the organs which are

losing it to those which are gaining it, at least partof it is synthesized anew and accumulates in thegrowing parts, for the amount of glycoside which isgained by the roots and shoots separately is greaterthan the sum of the amounts lost by the hypocotyland cotyledons over the period of the experiment.Figures for dry weights (Table 3) show that in thecourse of this experiment the cotyledons and

Biochem. 1952, 50

Wet wt.(g.)8-9

10-710-911-98-26-4

Asperuloside(% of dry wt.)

4-84-84-85.34-85-3

The formation of the glycoside throughout theseasonwas followed. Batches of20 shootswere takenfor as long as the rapidly increasing size ofthe shootspermitted. After that the number was reduced. Theamount of glycoside per shoot gradually rises andthen falls sharply as the shoot becomes senescent.When different parts of the shoot were tested withreagentA it was found that asperuloside is present inlarge amounts in all parts of the very young leaf,with the possible exception of the epidermis andchlorenchyma. There is much less in old leaves andwhat they do contain is confined to the vasculartissues, showing that the decline, already observedin the ageing hypocotyl and cotyledons, also occursin the leaves.The manner in which the glycoside content of the

plant changes with age was shown more clearly bythe following experiments with growing shoots,which were cut up into successive nodes, defined asfollows. The leaves grow in whorls about the nodes.Each node and the internode above it developtogether so that their parts have the same averageage and may be taken as a unit composed of tissuesof approximately the same age. Dry weights were

determined on material from a similar shoot from21

I I I ~~~~I Il

A

B -

C..... , -

VoI. 50 321

A. R. TRIMthe same plant and a corresponding node. Theresults in Fig. 3 show that where rapid, exponentialgrowth is taking place there is a high, constant con-centration of asperuloside; after exponential growthhas ceased there is a sharp downward trend inasperuloside concentration towards a lower, slowlydrifting level.

33

2 -

< -'~-: I I0

0., iiIiI1I

0 1 2 3 4 5 6 7Node number

Fig. 3. The concentration of asperuloside in successivenodes of a shoot of G. aparine. Node 1 is the most apical.The black columns indicate the relative dry weights ofthenodes.

If the change in asperuloside concentration isconsidered in relation to the development of anindividual mesophyll cell the change from the pre-dominance of accumulation to the predominance ofutilization (or transport away from the leaf)

appears to be sharp and to coincide with the end ofvacuolation and cell expansion. In this case (and allthe others described in this paper) the change doesnot occur in a particular node, say 3 or 4, countingfrom the apex of the shoot downwards, but appearsto be at a hypothetical point betweennode 3 and 4 (inthis case). This is probably to be explained by thefact that all the cells in a particular node are not atthe same stage of development. In this case all thecells in node 4 have passed through the stage ofexponential growth; but while some of those innode 3 have also passed through that stage many ofthem are still in it. In this situation it is logical toexpect the asperuloside concentration in node 3 tobe on the higher level, although growth has alreadybegun to slow down.

It seemed that during the stage of exponentialgrowth the accumulation of asperuloside wasdirectly proportional to the increase in dry matter.This was tested by estimation of asperuloside inshoots of G. aparine over a period of 2 weeks. Theshoots were chosen so that the whole of the tissueswere well within the stage of exponential growth.They were picked at random from the experimentalplot and divided into three batches of 30.

Asperuloside was estimated in two batches andthe third was used for dry-weight determinations.The results in Table 5 show that over a period duringwhich the bulk of the average shoot increased nearlythreefold the amount of asperuloside remaineddirectly proportional to the fresh and dry weights.

Accumulation of a&peruloside in the fruit. Theaccumulation of the glycoside during the period ofthe swelling of the fruit was also observed. Fruitwas collected at three successive stages. As isshown in Table 6, the amount of glucoside isapproximately proportional to the dry weight.

Table 5. Proportion of asperuIoside in the 8hoots of Galium aparine in the stage of exponential growth

Samplelabc

2abc

3ab

4abc

Date ofpicking14. iv. 47

18. iv. 47

22. iv. 47

29. iv. 47

Wet wt.(g.)10-512-412-412-312-512-114-816-427-829-231-1

Dry wt.(g.)1-331-571-581-541-571.511-671-843-63-840

Dry matter(%)

12-7

12-5

11-3

12-9

Asperuloside(% of dry wt.)

3-253-35

3-253-35

3-20

3-203-30

Table 6. Asperuloside in the fruit of Galium aparine

Condition of fruitSmall and softBeginning to hardenHardened

Wet wt. of30 fruit0-7660-9920 977

Dry matter(%)313337

Asperulosideper fruit(mg.)0-250390-44

Asperuloside(% of dry wt.)

3-23-73-6

322 I952

5ASPERULOSIDE

Table 7. Asperuloside in the leaves of Rubia tinctorum

Whorlno.123456

Length ofNo. of internodeleaves (cm.)

6 2-66 706 8-07 7*06 7.56 6-5

Dry matterper leaf

(g.)0 0030.0150 054005500450047

Asperuloside(% of dry wt.)

10*410-88-4503-72*4

Table 8. Asperuloside in the phloem of Rubia tinctorum

Wet wt. Dry wt. Dry matter AsperulosideStrip (mg.) (mg.) (%) (% of dry wt.)

1 12-0 2-35 - 232 10-0 1.95 243 13*3 2-60 19.54 10-2 2-0 19-5

Rubia tinctorumDeterminations of asperuloside in the growing

shoots of the dyer's madder, R. tinctorum, showedthat the same process of accumulation and sub-sequent utilization of asperuloside occurs in this

12

in_ -v

04.

3>.8

L.

vb

6 4

o3 5 7 9 11 13 15

Internode numberFig. 4. The concentration of asperuloside in successive

internodes of a shoot of R. tinctorum. Node 1 is the mostapical.

plant. In this case the initial concentration ofglycoside in the young leaves was much higher(10-15 % of the dry weight) than in Galium aparine.Figures for successive whorls of leaves, numberedfrom the apex ofthe shoot downwards, are tabulatedin Table 7. The length of the accompanying inter-node is given as an additional index of the stage ofgrowth.The values for the asperuloside concentration

in successive internodes of a developing shoot,numbered from the apex downwards, are recordedin Fig. 4. The arrow in this figure marks the ninthinternode below which the asperuloside concentra-tion rises. Comparison of these intemodes with

higher ones is scarcely permissible, since they hadbegun to develop characteristics of the subter-ranean parts of the plant. This was indicated by thegrowth of adventitious rootlets from some of thenodes and the appearance of anthraquinone glyco-sides, which are characteristic of the roots andunderground stems of Rubia. The observations inthe following section relate to the form of the curvein Fig. 4.

Asperuloside in the vascular tissues of the stem.A fully developed internode of R. tinctorum wasdivided into three parts: cortex, inner bark orphloem and xylem combined with pith. Testsshowed that there is scarcely any asperuloside in thecortex and woody regions, but there is a very highconcentration in the phloem. Fresh strips of thelatter were weighed rapidly on a torsion balance,dropped into a known volume of reagent A and theasperuloside estimated. Other strips were taken fordry-weight estimations. More than 20% of the drymatter was found to be asperuloside. The values arerecorded in Table 8. If younger, still growing inter-nodes are tested much asperuloside is found in thecortex and pith.

Asperula odorataThe pattern of distribution of asperuloside in

A. odorata was found to be very similar to that in thetwo plants already described. An experiment withthis plant was devised to confirm the observationsalready made. It was arranged to cover the periodof flowering to see if the development of the in-florescence is accompanied by asperuloside accumu-lation. The experiment was started when it was justpossible to separate the developing flower budsfrom the shoot and continued until the flowers werejust beginning to die. Sixty shoots were picked atrandom from one large clone at intervals of 3 daysover a period of 4 weeks. These batches were dividedinto two equal parts and the nodes of each- part

21-2

-0

0

itI I I I I

Vol. 50 323

I

I F

A. R. TRIM

separated. The leaves were separated from thestems and the inflorescence was counted as one node,number 1. The parts from one batch of 30 shootswere used for dry-weight determinations and thosefrom the second for asperuloside determinations.In this way it was possible to follow the course ofasperuloside accumulation and disappearance ineach unit. The results recorded in Figs. 5a and b

L.

8 12 16 20 24 28Time (days)(a)

cells appears to be as follows: after the primarymeristematic stage, the cells enter the period ofexponential growth, during which the glycosideaccumulates in amounts directly proportional to theincrease in dry matter. This may be the result ofsimple storage of the product but may equally bedue to the accumulation of glycoside, resulting fromthe balance between opposed processes of synthesis

8 12 16Time (days)

(b)Fig. 5 (a, b). The concentration of asperuloside in components of successive nodes of shoots of A8perula odorata during

flowering. The nodes are numbered from the apex downwards: (a) leaves, number 1 is the inflorescence; (b) inter-nodes, number 1 corresponds to leaves number 2.

show that the parts of A. odorata accumulate andutilize asperuloside according to the same develop-mental pattern as the two plants already investi-gated. In the inflorescence the glycoside level re-mains high throughout the experiment. However,this was the only part which continued to form newelements, thus compensating for the loss ofglycosidein the senescent elements.

DISCUSSION

While the study of individual cells has not beenpossible in this work, it appears that many of themaccumulate asperuloside during an early stage oftheir existence. In this respect the history of such

and removal, in which there is a preponderance ofsynthesis over removal. The disappearance of theglycoside could occur by further metabolism at ornear to the site of formation, by translocation, or bya combination of both processes. At the end ofexponential growth of the cells increase in volumevirtually ceases and a decline in glycoside accumula-tion sets in, so that finally the cells become devoidof it. This change would be explained either by acessation of synthesis or by a decline of synthesissufficient to be completely overbalanced by acontinued steady removal of glycoside, or by anincreased rate of removal. Whatever the explana-tion, the cessation of growth is accompanied by anabrupt change in glycoside metabolism.

324 I952

ASPERULOSIDEThe chlorenchyma may not accumulate asper-

uloside and the epidermal cells also appear to beexceptional in that very few, if any of them,accumulate the glycoside. Since the methods em-

ployed were crude these conclusions are onlytentative. In all organs part of the vascular systemappears to be exceptional in continuing to accumu-late asperuloside until the organ becomes moribund.The technique available did not permit a distinctionbetween individual cells, but it is certain that thisphenomenon is confined to the inner bark, that is, tothe phloem and cambium. But this is a region ofcontinued growth, containing young cells whichwould be expected to contain the maximum amountofasperuloside. Consequently, it might be suggestedthat the exceptional behaviour of these parts, inrespect of asperuloside accumulation, is more

apparent than real. However, it should be addedthat the amounts of asperuloside found in the innerbark were in excess of what might have been ex-

pected if it were composed entirely of very young

cells, which is not the case. In addition, it has to beremembered that the phloem is the main path oftransport for organic material, so that the largeamounts of asperuloside found there may be inprocess of transport.As far as the author is aware this is the fullest

investigation of its kind. But there are many otherrecorded observations which support the view thatthe distribution of asperuloside in the rubiaceousplant is typical of the distribution of glycosides andmany other metabolites throughout the wholerange of higher plants. It is frequently stated in theliterature that young, actively growing parts of a

particular plant are the most abundant source ofsome of its characteristic products. The young

shoots of many plants are pigmented by antho-cyanins in the developing epidermal cells, and thepigmentation often disappears after these cells havebecome fully grown. In his observations on theliving rose leaf, Guilliermond (1913) showed thatanthocyanins accumulate in the developing vacuolesof the young epidermal cells, and thus provide a

visible parallel to the first stage of the process whichhas been postulated to explain the present observa-tions on asperuloside. Hurry (1930) showed that thelargest amount of the indigo-producing glycoside ofI8atis tinctoria is present in the very young leavesand that the older leaves contain much less. Theclassical work ofTreub (1896, 1904, 1907) on cyano-

genesis in Pangium edule and other plants providesfurther examples of the same developmentalprocess.

Certain exceptional cases have been observed.Bourquelot & Danjou (1905), and others, have

shown that sambunigrin, D-mandelonitrile gluco-side, which occurs in the elder, Sambucus nigra,remains in the leaves even after they have fallen.Treub (1904) noticed the same phenomenon with thecyanogenetic glycoside of Indigofera galegoides.There is a suggestion in such cases that the appro-priate hydrolysing enzyme is absent, but it has yetto be shown that such phenomena are not alsodependent upon the absence of all of a number ofdifferent modes of utilization of the glycoside con-cerned. At the other extreme, Treub (1904) foundthat in Pha8eolus lunatus there is a marked diurnalvariation in the amount of cyanogenetic glycosidestored in the leaves.The balance of evidence clearly favours the view

that glycosides are rapidly synthesized and brokendown in the plant and that in some cases at leastlarge amounts of glycoside are involved. Thecharacteristic distribution of these substances in theplant is related chiefly to different metabolic stagesin its development. If, as it seems, glycosides enterinto the metabolic processes of plants, they cannotbe regarded as mere end products of metabolism,and it is important to determine the reactions inwhich they participate.

SUMMARY

1. The accumulation and utilization of asperulo-side in the tissues of three rubiaceous plants havebeen studied.

2. The glycoside accumulates in young tissuessuch asnew leaves in the stage ofexponential growthand cells in the neighbourhood of the cambium.

3. The accumulation stops abruptly with thecompletion of the stage ofexponential growth and isfollowed by a period of glycoside depletion, whichmay involve translocation to another part of theplant.

4. Nearly all the cells in the plant, with thepossible exception of most of those in the epidermisand chlorenchyma of the aerial parts and some ofthose in the roots, appear to accumulate asperulosidewhen in the early stage of growth.

5. The significance of these results in relation tometabolism and translocation is discussed.

6. The general pattern of production and utiliza-tion of asperuloside appears to be characteristic ofmany glycosides and other metabolic productsthroughout the higher plants.

The author wishes to thank Dr R. Hill, for his closeinterest and advice, also Dr A. C. Chibnall, F.R.S., andDr C. S. Hanes, F.R.S., for their interest in this work, whichwas carried out as part ofthe programme ofthe AgriculturalResearch Council Unit of Plant Biochemistry, Cambridge.

325V5is. 50

326 A. R. TRIM I952

REFERENCES

Bourquelot, E. & Danjou, E. (1905). C.R. Acad. Sci., Paris,141, 59.

Guilliermond, A. (1913). C.R. Acad. Sci., Pari, 157, 1000.Hurry, J. B. (1930). The Woad Plant and its Dye, p. 43.

Oxford: University Press.

Treub, M. (1896). Ann. Jard. bot. Buitenz. 13, 1.Treub, M. (1904). Ann. Jard. bot. Buitenz. 19, 86.Treub, M. (1907). Ann. Jard. bot. Buitenz. 21, 79.Trim, A. R. & Hill, R. (1952). Biochem. J. 50, 310.

A Modified Medium for Lactobacillus casei for the Assay of B Vitamins

BY K. M. CLEGG, E. KODICEK AND S. P. MISTRYDunn Nutritional Laboratory, Univer8ity of Cambridge and Medical Re8earch Council

(Received 9 March 1951)

Snell & Wright (1941) were the first to point outthat their method for the assay of nicotinic acidcould be applied to the determination of biotin andpantothenic acid. Shortly afterwards, Landy &Dicken (1942) published a general procedure for theestimation of six vitamins, using Lactobacillus caaeias the test organism. Roberts & Snell (1946)devised a medium for microbiological assays withL. ca8ei, which was an improvement on the originalmedium of Landy & Dicken (1942).On investigating the various factors affecting the

growth of L. ca8ei, we have modified the medium ofTeply & Elvehjem (1945) with the aim ofdevelopinga suitable medium which could be used for assays ofvarious vitamins.

MATERIALS AND METHODS

Stock 8OlUtiOfl8Unless otherwise mentioned, the stock solutions were pre-pared according to techniques described by Snell (1950).

Salt 8olution E (Kodicek & Pepper, 1948a).MgSO4. 7H20, 60 g.; FeSO4. 7H20, 4 mg.;MnSO4.4H20, 1-5 g.; CuSO4 .5H20, 4 mg.;NaCl, 0-5 g.; ZnSO4. 7H20, 4 mg.

were dissolved in 250 ml. of water and 3 drops of conc. HCIadded.

Ca8einhydroly8ate:H2$O4-hydroly8ed,vitamin-free'Labco'ca8ein. 'Labco' casein (200 g.) with 1 1. 25% (w/w) H2S04,was autoclaved for 15 hr., at 15 lb. pressure. A litre ofwater was added, sulphate precipitated with about 850 g.Ba(OH)2, the mixture autoclaved for 10 min. at 10 lb.pressure, and filtered hot through a Buchner funnel. TheBaSO4 cake was thoroughly broken up in 1 1. water, auto-claved and filtered hot. The precipitate was washed oncemore as before. The combined filtrates were heated and theexcess barium was carefully precipitated with 25% H2504.The suspensionwas filtered hot, andthe filtrate concentratedonawater bath to about 2500 ml. Dryweight determinationgave a recovery of a little over 90 %.The hydrolysate at pH 6-0 was stirred for 1 hr. with

100 g. Norite charcoal (British Drug Houses Ltd.), filtered

and the pH adjusted to 3-8 with glacial acetic acid. Thecharcoal treatment was repeated, the solution filtered andbroughttopH 6 8-7-0 with40% KOH. The hydrolysate wasfinally treated with Norite, 40 g., for 1 hr. The residue fromeach ifitration was washed with 50-100 ml. of water and thewashings added to the filtrate. The filtrate was concentratedon a water bath to contain 10% (wfv) casein solids. Re-covery was 73% (casein solids). Before use, new batches ofhydrolysate were always tested at several levels and theoptimum concentration determined. An amount of 50 mg./10 ml. medium was usually found adequate. We have foundit necessary to restandardize the adsorption treatment whena different brand of charcoal was used.

Peptone treated with Norite charcoal. 'Difco' bacto-peptone (1O g.) was dissolved in about 80 ml. water, pHadjusted to 3 0 with HCI and made up to 100 ml. Thesolution was stirredwith 5 g. Norite for 1 hr., ffltered, andthecharcoal treatment repeated twice with 2 g. Norite.

Pteroylglutamic acid (PGA). During the early part of thework 'Folvite' (Lederle Laboratories) was used as the PGAstandard. Later, when a 97% pure and aldehyde-free pre-paration (Lederle Laboratories) became available it wasused instead.A stock solution of PGA containing 100,ug./ml. was

prepared in 1% K2HPO4. It was renewed twice weekly.From the stock solution weaker standards were prepared forimmediate use.

Bacteriological procedureThe double-strength medium, 5 ml., the composition of

which is shown in Table 1, was diluted to 10 ml. with distilledwater after adding the test solutions. The test substanceswere assayed usually in triplicate at each level. The tubeswere placed in Petri dish canisters and autoclaved for 6 min.at 15 lb. pressure. Autoclavingfora longer period resulted incaramelization and consequent deterioration ofthe medium.On autoclaving, a slight precipitate resulted, but this re-dissolved on shaking at room temperature.

Stock culture. Cultures ofL. casei, A.T.C.C. no. 7469, withcharacteristics reported by Kodicek & Pepper (1948b) weremaintained on slopes of' Difco' yeast-dextrose-agar supple-mented with 'Hepamino' liver powder (Evans) 1% andglucose 0.5%. They were subcultured every 2-3 weeks.Each subculture consisted of two transfers from liquid to