THE STABILITY 1~~0s
attempts at hydrogen SUCH tracts as have been made
ion concentration control of pollen exin the past have been directed toward buffering the solution at an alkalinity approximately that of body fluids. No direct comparison has been made of the initial potency and the stability of pollen extracts in various buffer solutions, acid and alkaline, under the same conditions. Stier and Hollisterl have reported that addition of sodium bicarbonate decreases the antigenic properties of pollen extracts. The purpose of this study has been to determine whether slight acidity or alkalinity, as produced by buffers, has any effect on the potency and stability of pollen extract,s. All other factors have been kept constant so far as possible. Experiments conducted over three years have demonstrated that pollen extracts in 5 per cent dextrose are as stable as those in the commonly used glycerosaline.2 The present work has been carried out by comparison of the buffered extracts with an unbuffered extract in dextrose menstruum. EXPERIMEKTAL
The buffer solutions used as extracting fluids were prepared by adding to each 90 cc. dextrose menstruum, the following buffer solutions3: II. 9.5 cc. KH,PO, III. 3.0 cc. KH,PO, IV. 0.5 cc. KH,PO,
(M/15) (M/15) (M/15)
and 0.5 cc. Na,HPO, and 7.0 cc. Na,HPO., and 9.5 cc. Na,HPO,
V. 6.75 C.C. NaHCO, (2.7%) Solut,ion I, unbuffered dextrose menstruum, cent dextrose and 0.5 per cent phenol.
(M/15) (M/15) (M/15) 5 per
Pollen extracts were prepared by extracting 3 gm. pollen with 100 cc. of t,he extracting fluid. Equal parts of short and giant ragweed pollen were used. the
Table I shows the hydrogen ion concentration of the solutions and of the finished extracts prepared from them, as determined by the glass The buffering capacity of the pollen itself appears quite electrode.* remarkable. TABLE -. -
4.2 5.6 7.1 7.6 8.3
II III IV v
EXTRACT ti.4 6.1 6.6 67 7:s
The freshly prepared extracts were tested by intradcrmal comparisons Similar extracts of serial dilutions upon ragweed sensit,ive patients. were placed in the incubat.or at 37” C. to speed up any deterioration, and were then compared by the same method after incubation for thirteen months or longer. For testing, decimal dilutions of the extracts were prepared from l:l,OOO up to .I:1,000,000,000. The proper dilutions were injected intradermally into sensitive patients, taking cart t,o inject the same amount in each case. Because of lack of space, all the solut,ions could not be compared upon the same patient at t,he same time, but at, ea.ch sitting two to four of the buffered ext,racts were compared with the unbuffered one. The aim was to find the dilution at which the extract just failed t,o react. The cases in which this goal was not attained, due to improper selection of dilutions for the patient,, arc not included in t,he reports. The extract reacting in higher dilution was assumed to be the stronger. In order to avoid undue length of tables, only the final results are recorded. Table II shows the results of comparative t&s of the freshly prepared extracts. 11
No. No. No.
of of of
tests tests tests
stronger than I equal to I weaker than I
1 1 ”*, 9
UNBT:FFF,RED _____ III 1: 7 13
3 10 8
2 12 8
The number of tests, as given in Table II, refers to the number of patients upon whom the given buffered extract reacted in higher, equal, or lower dilution than the unbuffered extract. TABLE COSXPARISON
_ No. No. No. *We
of tests of tests of tests indebted
stronger than I equal to I weaker than T to Mr.
K. J. Bauer
7 9 0 for
5 5 5 determinations.
AGED IV 7 3 1
EXTRACTS v 14 4 2
After the samples had remained in the incubator for thirteen months, dilutions were made and their relative ljotency was determined by comparative intradermal tests on hay fever patient-s by the same methods used for the fresh extracts. TAater dilutions were made after longer incubation, but in eveqr cast all samples had rcmaincd in the incubator for the same length of time and ilr(l. thcrcforc~, romparablc. Table IIT gives a sumnlilry of these.
Buffered extracts of the composition of (V) have been used for t,rcatmerit of hay fever patients for about six months. The buffered and unbuffered est,racts wcr~ freely int,crchangcd with no clinical difficulty, e.g., reactions. The LISC of the hnffc~rcd casirwct was not. attended b> pain such as comes with glyccrinatcd extracts. The results of treatment likewise 30 not stem to differ. DISCUSSIOP\‘
While the differences above noted are not marked, it may- be stated that freshly prepared pollen extracts in unbuffered isotonic dextrose solution are at least as potent as those prepared in solutions buffered at a point slightly acid or slight.ly alkaline. There is some evidence that buffering at cithcr slightly acid or alkaline hydrogen ion concentration may incrcasc the stability. The extract buffered with sodium bicarbonalc (T’) has the advantage that no glucoside is deposited on st,anding, as occurs with all the other solutions. The quantity of extract afttr incubation did not permit a determination of the hydrogen ion oollcclltri~tio11, but, it probably had become more. alkaline t,han hydrogen ion conrcntration 7.5, due to loss of carbon dioxide. The color of alkaline extracts is very much deeper than that of unbuffered extracts. SUMMARY
It has been shown that the presence of buffer salts in dextrose menstruum dots not, increase the potency of ragweed pollen extracts prcpared with such solutions. Ragweed pollen extracts in isotonic dextrose buffered at either a slightly acid (hydrogen ion concentration 6.1) or slightly alkaline (hydrogen ion concentration 7.5) reaction have been shown to bc at least as stable as similar unbuffered extracts. There is some indication that such buffering may incrcasc stability. Alkaline buffer is probably to bc preferred, as it prcvcnts precipitation of the glucoside and thus prevents the development of a cloudy appearance on standing. Therapeutically, the same results were obtained with the extract buffered with sodium bicarbonate as with a similar unbuffered extract.
REFERENCES 1. Stier,
F. E., and Hollister, G. L.: ii Comparatire Study of Pollen Antigens as Determined by the Skin Reaction, J. Lab. & Clin. Med. 12: 1139, 1927. 2. Unger, L., and Moore, M. B.: IX. A New EMracting Solution, J. ALLERGY 4: 92, 1933. XIII. Dextrose Pollen Extracts: Therapeutic Results in 1933, J. ALLERGY 5: 561, 1934. 3. Clark, W. AI.: The Determination of Hydrogen Ions, ed. 3, Baltimore, 1925. p. 210, Williams $ Wilkins Co.