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L. H. SOPHIAN .4ND V. J. CONNOLLY
(5) HARTLEY, G. SPENCER: J. Chem. SOC.1938,1968.
(6) HOLMES,HARRYN.: J . Phys. Chem. 48,495 (1939). (7) KLEVENS, H. B . : J . Chem. Phys. 17, 1004 (1949). (8) KOLTHOFF, I. M., AND JOHNSON, W. F . : J. Phys. Chem. 60,440 (1948). I. M., AND STRICEP, W . : J. Phys. &Colloid Chem. 62, 915 (1948). (9) KOLTHOFF,
(10) KOLTAOFF, I . M., AND STRICKS, W . : J. Phys. & Colloid Chem. 68, 424 (1949). Kolloid-Z. 98.45 (1942). (11) LAMM,0.: R . S., AND HARKING, W. D.: J. Chem. Phys. 16,644 (1948). (12) MATTOON, R . W., STEARNS, R . C . , AND VINOGRAD, J. R . : J. Am. Chem. SOC. 63. 670 (13) MCBAIN,J. W., MERRILL, (1941). (14) MCBAIN,JAMESW., WILDER,ARTHUR G . , AND MERRILL, R. C., JR.:J. Phys. &Colloid Chem. 62, 12 (1948). (15) RICHARDS, PAULH . , AND AICBAIX,JAMESW.: J. Am. Chem. SOC.70,1338 (1948). (16) SMITH,E. LESTER:J. Phys. Chem. S6, 1401 (1932).
ULTRAVIOLET ABSORPTION SPECTRA OF NORMAL 'CEREBROSPINAL FLUID LAWRENCE H. SOPHIAK
AND
VALENTINE J. COKNOLLY
U. S. Marine Hospital, Staten Island 4, New York Received June 86, 1960
Conflicting reports concerning the ultraviolet absorption spectra of cerebrospinal fluid can be found in the literature. Thus Spiegel and coworkers (3) reported normal cerebrospinal fluid as possessing a weak and characterless S-shaped curve. They also indicated that some specimens obtained from patients suffering from pathological cerebral conditions differed from normals by showing an absorption band in the region of 265 mp. This absorption was considered (4)to be related to nucleic acid substances liberated by cell injury. Subsequently Strait (5) reported that the absorption band a t 265 mp was present in all cerebrospinal fluids and that the normal ascorbic acid content was the substance responsible for the absorption in this region. Our investigations were made while conducting a search for atypical ultraviolet absorption spectra in cerebrospinal fluids. The specimens were obtained mainly during the course of the preoperative administration of intraspinal anesthesia to patients undergoing surgical operations. The largest percentage were being operated upon for hernia and presented no pertinent abnormal findings. The Beckman DU spectrophotometer, with the necessary attachments for studies in the ultraviolet region, was employed. In order to avoid the alteration in the shape of the ultraviolet absorption curve when albumin is treated with acids and alkalies (2), aqueous dilutions were used. All spinal fluids were studied in 1:10 dilution of the specimen with distilled water. A 1-cm. cell was used and readings were taken a t IO-mp intervals until the absorption region was approached, where the readings were taken at intervals of 5 mp.
ABSORPTION SPECTRA OF CEREBROSPLVU FLUID
713
I n figure 1, line A shows the absorption curve obtained using a fresh aqueous solution of ascorbic acid in a concentration of 4.0 mg. per 100 ml. Before making the spectrophotometric readings this concentration was further diluted 1:10, duplicating the procedure used for all spinal fluid specimens. Line B of the same figure demonstrates the curve obtained from a fresh solution of human serum albumin in a concentration of 50 mg. per 100 ml. These concentrations were chosen to approximate the normal ranges usually reported for these substances in lumbar spinal fluid. Line C illustrates the curve obtained using a mixture of the two substances in the same final concentration.
IO
.
A
B c
-0-
ASCW C I
- AWMIN
IO
.
A 0
-0-
ASCORSIC ACID
-ALBUMIN
--- ASCORBICALBUMIN C -- Areoser ALWMIN n o m n o m m z m m ~ m ZH) a0 ldo m 20 uon FIQ.1 FIQ.2 FIG.1. Readings on pure solutions a t zero time interval FIG.2. Readings on pure solutions a t 3-hr. intervals
As expected, the absorption bands of ascorbic acid and albumin Then tested separately in pure solution are as one would anticipate a t 265 mp and 280 mp, respectively. However, in the mixture, the absorption due to the ascorbic acid overwhelms the albumin concentration to the extent of completely masking the band at 280 mp. The additive effect of the separate absorptions is obvious. Figure 2 shows the curves obtained from the same solutions after they had been allowed to remain at room temperature for a period of 3 hr. In the pure solutions the rapid oxidation of the ascorbic acid is readily seen, while the absorption of the albumin is relatively unaltered in this time interval. In the mixture the oxidation of the ascorbic acid now enables one to observe the absorption band a t 280 mp due to the albumin present. Figure 3 represents the observations on the same samples after they had been allowed to remain at room temperature for a period of 48 hr. One sees that in this time interval some modification of the structure of albumin has taken place, as evidenced by the disappearance of the absorption band at 280 mp. Line A in figure 4 shows the results obtained on a typical sample of normal
714
L.
n.
SOPHIAN AND
v.
J. CONNOLLY
cerebrospinal fluid. In more than 90 per cent of our samples of spinal fluid this is the absorption curve found when the material is examined immediately. A comparison of these curves with those obtained from the mixture of fresh ascorbic acid and albumin in the same relative concentrations shows the striking similarity 0' both the shape and the intensity of the spectrum between 220 mp and 320 m,u. One may, therefore, conclude that the content of ascorbic acid and albumin can account for the character and intensity of the ultraviolet absorption spectrum of fresh normal cerebrospinal fluid. The influence of time on the shape and intensity of the curve is illustrated in lines B and C. We noted that the oxidative changes were of such rapidity that a delay of as little as 5 min. significantly influenced the results. The weakly S-shaped curve resulting follofifI
/
10
1, l
ALBUMIN
B-
c - - Ascqeu ALBUMIN MIXT.
/ .
230
140
.
250
.
260
.
270
.
280
. 1
2(10 mu
FRESH
A-
B
iul'
c
nu
140
To
3 now
--- 48WURJ -0-
w
zm ao mn
FIG.3 FIQ.4 FIG.3. Readings o n pure solutions at 48-hr. intervals FIi;.
4 . Readings on rercIrospi~i:ilfluids a t different time intervals
ing the oxidation of the ascorbic: acid and the degradation of the albumin probably indicates the appearance of breakdown products. It is clear that reliable absorption spectra in the ultraviolet can only be obtained on fresh cerebrospinal fluid. Observations should also be made after certain time intervals to note the disappearance of some substances and the appearance of other absorption bands not evident on immediate esamination. Studies were made to determine whether the characteristic absorption band for ascorbic acid a t 265 mp could be utilized for the quantitative estimation of the substance. Simultaneous chemical determinations of ascorbic acid were run, using the colorimetric procedure of Mindlin (1). A calibration curve for the absorption of different concentrations of ascorbic acid at 265 mp was obtained, and this was used to convert the readings of the samples to quantitative terms. The concentrations obtained in the spinal fluid by using the direct reading of the spectrophotometer were always higher than the chemical method but there esisted no constant percentage difference. This can be attributed to the additive
715
ABSORPTION SPECTRA OF CEREBROSPINAL FLUID
influence of spinal fluid protein on the absorption a t 265 mp, as was demonstrated by using pure solutions. A pure solution of ascorbic acid gravimetrically calculated to be 3.8 mg. per cent read exactly that when tested both chemically and spectrophotometrically. However, when the same solution was tested after the addition of albumin to give a final concentration of 50 mg. per cent, the result obtained by the 2,6-dichlorophenolindophenolreaction was still 3.8 mg. per cent, while that of the ultraviolet absorption reading gave a result of 4.5 mg. per cent. We investigated the possibility of obtaining the concentration of the ascorbic acid by the difference in absorption at 265 mp of the fresh specimen and that of the same specimen after allowing sufficient time for complete oxidation of the ascorbic acid present. This principle is that suggested by Strait ( 5 ) , who recommended the measurement of the difference in extinction before and after alkaline destruction of ascorbic acid a t pH 11. Our findings indicated that while this technique resulted in closer correlation with the chemical findings than did the direct reading at 265 mp, the results often showed wide disagreement. The results by the absorption method were again higher than those obtained by the chemical technique. A sample which demonstrated 1.5 mg. per cent chemically resulted in 2.2 mg. per cent using the absorption readings. We further attempted to correlate our findings of the quantitative estimation of ascorbic acid by the chemical technique with the results obtained using the absorption a t 265 mp before and after alkaline destruction of the ascorbic acid. Once again we found that the results obtained spectrophotometrically were higher than those obtained colorimetrically. When a pure solution calculated to be 3.7 mg. per cent ascorbic acid was tested, B value of 3.6 mg. per cent was obtained by use of the alkaline destruction technique. When the same concentration of ascorbic acid was tested following the addition of albumin to give a concentration of 50 mg. per cent, the resultant absorption reading was equivalent to 4.3 mg. per cent ascorbic acid. The presence of albumin apparently does not influence the results of the colorimetric estimation of ascorbic acid but does interfere with the absorption techniques even though alkaline destruction of the ascorbic acid is applied. Determinations on cerebrospinal fluids demonstrated the same discrepancy betwren the chemical and the spectrophotometric estimation of ascorbic acid. Typical results obtained simultaneously on identical specimens are illustrated below:
~mg.
1 2
3
,
per ccnl
1.9 2.1 3.2
~
I
mg. per cent
1.0 1.5 2.5
SUMMARY
A study was made of the ultraviolet spectral absorption of pure solutions of albumin and ascorbic acid in the range of concentration commonly found in
716
GIUSEPPE CILENTO
cerebrospinal fluid. The influence of time upon the shape and intensity of the curve was noted. The curve obtained on these pure solutions was compared with that obtained on normal cerebrospinal fluid under the same conditions. Comparison of the quantitative estimation of the ascorbic acid content of the fluids by the ultraviolet absorption and by the 2,6-dichlorophenolindophenol colorimetric reaction consistently showed a false elevation by the former methods. REFERENCES (1) NINDLIN, R.L . , A N D BUTLER,4.M.:J. Biol. Chetn. 122, 673 (1938). (2) SIZER,I . , A N D PEACOCK, A.: J . Biol. Chem. 171, 767-77 (1947). (3) SPIEGEL, E . A , , SPIEOEL-ADOLF, hi., A N D WYCIS,R. T . :Science 106, 208 (1947). (4) SPIEOEL-ADOLF, M.,A N D WYCIS,H . : J . Phys. Chern. SO, 447-52 (1946). ( 5 ) STRAIT, L.A., AIRD,R. B . , A X D HRENOFF, M. K . : Science 106. 64-5 (1947).
BISARY SYSTEMS BETWEES PHEPI'YL BENZOATE DERIVATIVES
THEFORMATION OF MOLECULAR COMPOUNDSI GIUSEPPE CILENTO'
Deparlainento de Quiinica, Facitldade de Filosofia, Citncias e Letras, L'niversidade de Sdo Paulo, Sdo Paulo, Brasil Received M a y Si, 1960
In 19-11 H. Lettrb (1) reported that nitrobenzoic acids very often form a 1:l molecular compound with unsubstituted or substituted benzoic acids. He attributed this result to the presence of the nitro group, since compound formation was not observed with nonnitrated benzoic acid. Furthermore, he stated that the carboxyl group should also be involved in compound formation, since the corresponding phenyl esters display no tendency for molecular addition. During research upon the relationships between the structure and the isomorphism of organic compounds, we have found that in a few cmes even esters, closely related t o those investigated by LettrB, can form molecular compounds although in a 2: 1 molar ratio. The purpose of this paper is to report the corresponding solid-liquid phase diagrams. Information is also given for related systems, which were studied in order to ascertain the degree of specificity for the formation of molecular compounds. Figure 1 shows that p-tolyl p-nitrobenzoate forms a molecular compound with 1 P u t of the thesis submitted by Giuseppe Cilento in partial fulEllment of the require. ments for the degree of Doctor em CiBncins at the Universidade de SLo Paulo, Brasil, October, 1916. 2 Present address' FundnC5o Andr6a e Virginia Matnrszzo, Fsculdnde de Medicins, 'Universidnde de SBo Paulo, Cniw Postal 100-B, Sgo Paulo, Brasil.
