Magnetic circular dichroism spectroscopy

ANALYTICAL

BIOCHEMISTRY

Magnetic A Definitive SU?tINER

Method

52, 83-95

(1973)

Circular

Dichroism

for the Determination

31. KALMAIS, GiiKTER EDWARD BUKSENBERG,

Kecrived

Spectroscopy

June

26, 1972;

of Urinary

Porphyrins1

BARTH, ROBERT E. LISDER, ASD CARL DJERASSI

accepted

September

14. 1972

The application of MCD for the determination of urinary porphyrins is assayed by specific application to the screening of workers exposed to lead arsenate. The definitive nature of this new spectroscopic method arises from: (1) its sensitivity to as little as 0.02 &ml of porphyrin dication; (2) its frerdom from interference caused hy other substances: (3) the highly characteristic shape of the MCD bands exhibited by porphyrin dications; and (4) the high information content of the MCD spectrum.

That lead poses a threat to man’s well-being was yiviclly recorded by Nicander of Colopl~on over 2,000 yr ago Cl). Since the industrial revolution, the increased use of lead and its derivatives in expanding economies has provided ample opportunity to assess the effects of exposure to lead in a wide variety of forms and on groups of increasing statistical significance. Furthermore, comparison of the usual daily intake of adults, which in the United States averages 0.3 mg, with the 1-mg level of sustained daily intake which has been estimated to mark the level at which stimulated metabolic response becomes evident (21, suggests that. a group large enough to be statistically definitive will be available in the near future. Indeed, from the viewpoint of some investigators such a definitke group is already storing potentially dangerous amounts of lead in their body t.issues (3,4). The pervasive presence of lead in the environment, its ability to accumulate in tissues, and the serious health hazards result’ing from lead poisoning, especially in the case of children for whom brain damage is common and learning difficulties cert’ain, places high priority on effective pollution control measures (5,6) and on the development of techniques to aid in its diagnosis. It’ is in this respect that plumbism acquires its in‘Paper Studies.”

XXII in the Stanford University Series For the preceding part. SW Ref. 16. 83 Copyright @ 1973 by Academic Press. Inc. All rights of reproduction in any form reserved.

“?vlagnetic

Circular

Dichl~oiam

84

KALMAN

ET

AL.

sidious character; its overt symptoms are sufficiently varied, non-specific, and mild as to make diagnosis extremely difficult in the absence of specific suspicion. Recent studies of lead-intoxicated children indicate that prior “cut-off points” for blood lead levels may be too high, and that even moderate elevation of lead in blood or urine may indicate exposure to amounts of lead that can lead to brain damage in the absence of classical symptomatology. Thus, analytical indicators are mandatory although, unfortunately, none of the methods at hand is entirely satisfactory. Blood and urinary lead determinations using either t’he dithiozone color reaction (7) or atomic absorption spectroscopy (8) are of uncertain value owing to the storage factor as well as to the rapid decrease in lead levels with time. Other procedures have been developed for substances whose increased levels are indicative of lead intoxication, In particular, the finding of increased levels of coproporphyrin and porphyrinogen precursor, delta-aminolevulinic acid (ALA) and porphobilinogen in the urine and above normal amounts of protoporphyrin in whole blood serve as somewhat more reliable indicators. Both absorption spectrophotometric (9) and spectrophotofluorometric (10) procedures have been reported for urinary porphyrins. A highly sensitive fluorimeter has also recently been developed for determining porphyrins in blood, urine, and stools and is said (11) to be of particular value in mass screenings, especially for protoporphyrin in blood. In the present communication, we report the results of a preliminary evaluation of the utility of magnetic circular dichroism (MCDl for the determination of urinary porphyrins. The application of this new method to the screening of workers exposed to lead arsenate provides an appropriate data base for comparison with other technicpies. METHODS

AND

PROCEDURES

(a) The Group Sampled The group chosen for this study comprised the entire staff of 38 persons employed by a well-established tree-care firm in the San Francisco Bay Area, and included sprayers, helpers, tree surgeons and office personnel. The group was particularly germane for evaluating the utility of MCD for determining moderately elevated levels of urinary porphyrins thereby signaling possible cases of low level lead intoxication. This possibility arose because the actual spraying season in the spring of 1971 was only 1 mo long. Lead arsenate spraying operations were carried out under t,he direction of a team foreman. The foreman’s direct involvement in all phases of the operation from the preparation of solutions containing 0.1% lead

DETLR~lISATION

OF

PORPHYRIIiS

BY

85

MCD

arsenate (by weight I by mechanical mixing of bulk quantities of the dry powder’ to the actual spraying operation itself. The lead arsenate solution was applied as a fog from high pressure pumps at the rate of approximately 30 gal/min. The mechanism of exposure was primarily through inhalation of the dry powder during preparation of the solution and through inhalation of the fog. The latter source probably accounted for the major intake since the filters of the face masks provided to each worker were said to cause difficult’y in respiration when wet and were, in fact, rarely used. Absorption from wet clothing probably constituted a relatively minor risk as compared to inhalation. (bi The Sampling Most of the tests were performed just at the end of the month-long spraying period. Hair samples were collected for arsenic analysis and these results will be reported elsewhere. Four urine samples were collected, the first, being a ‘[morning sample.” The other two 24-hr samples were collected in the second and fourth weeks following the first sample collection. The samples were prot,ected from light. and stored at 4” prior to measurement. The samples were coded so as to preclude bias on the part of the analyst. A selected set of samples of 100 ml each from a 24-hr specimen was sent to Bio-Science Laboratories, Van Nuys, California, for independent porphyrin analysis. (c) The Analyses Urinary

Porphyrins

by MCD

Spectroscopy

The basis of the analysis. Magneto-optical activity is a universal property of matter and can readily be measured using the same instruments that are in common use for measuring natural optical activity. For reasons that are given elsewhere (12), circular dichroism is the preferred technique. The only modificat.ion required is that provision be made for immersing the sample in a magnetic field oriented such that the field is parallel to the propagation direction of the beam of circularly polarized light. Since JICD is linear in the applied field, superconducting magnets, capable of producing fields of 5&100 or more kilogauss (kG), are commonly used. The static magnetic field can perturb the wave functions of the substance in one or more ways depending on the details of the electronic st,ructurc of the molcculc. The dication derivative of coproporphyrin, for ‘Supplied as “Standard Lead Arsenate” by stated to cont;Gn 05% PhH.-is04 and 5% “inert”

the Chevron filler.

Chemical

Company

and

86

KALMAN

ET AL.

example, possesses a highly symmetrical and planar conjugated rr-electron system. The effect of t’he magnetic field on x-systems having at least a three-fold rotational symmetry axis and a totally symmetric ground state is to lift degencracies in the excited states, referred to as Zeeman splitting. Under the condit’ions of the circular dichroism experiment, it is found that left circularly polarized light (LCPL) is effective in promoting an elcctronic jump to one but not t.he other of the split energy levels. For right circularly polarized light. (RCPL), the converse is observed. The resultant curve has a derivative shape with peak-to-trough amplitude proportional to the concentration. The MCD and absorption spectra of coproporphyrin tetramethyl ester dication obtained in 0.2 A$-trifluoroacetic acid-benzene solution arc shown in Fig. 1. The principle features in the absorption spectrum are attributed (13,14) to electronic transitions from a totally symmetric ground state to excited levels commonly labeled B, and Q,,, both of which arc of E,, symmetry and therefore degenerate in the D,, point group. In a magnetic field these states are no longer of the same energy but rather are raised and lowered by the same amount about t,hc zero-field absorption maximum. This splitting is especially difficult to observe in the broad-band absorption spectra exhibited by organic molecules in solution although direct Zeeman measurements on porphyrins using a 100 kG superconducting magnet have been reported (15). In MCD the task of evaluating the Zeeman splitting is much easier because of the selection rules referrcrl to

120

40

FIG. 1. MCD (--) and absorption (---) spectra ester dication in 0.1 N trifluoracetic acid in benzene.

of

eoproporphyrin

tetramethyl

DETERMINATION

OF PORPHYRINS

BY

87

MCD

in the preceding paragraph. In particular, the MCD curve of coproporphyrin tetramethyl ester dication (Fig. 1) shows that RCPL is selectively absorbed in the promotion of an electron to the lower energy Zeeman sublevel whereas LCPL is selected for the higher Zeeman sublevel. These statements are applicable to the A terms associated with the B,-, and Q”-, transitions but not to each of the vibrational components of the Q,,-, band system (16). For example, the A term centered at about 568 nm is of inverted sign as compared to that of the Q,-, band at 590 run (16,17). For comparison, the MCD and absorption spectra of uroporphyrin octamethyl ester are shown in Fig. 2. Although uroporphyrin is found in higher than normal amounts in the urine of lead-intoxicated patients, its level remains far below that of coproporphyrin (18). The application with which we are concerned is based on several observat,ions. First,, porphyrin dications exhibit exceedingly intense MCD bands, especially in the region of the Soret absorption band (400-410 nm). In practice, this means that very minute amounts of the dication are easily detected. In particular, as little as 0.02 PgJml of coproporphyrin dication can be readily determined !I-cm path length cell) without special low signal level procedures being required. In absorption, this sample would exhibit an absorbance of only 0.01 units and would thus escape detection on most, if not all inst,rumente. With this result in mind, a comparison of the ?1ICD and absorption spectra shown for the two dications in Figs. 1 and 2 leaves little doubt that 1\ICD is the method of choice. Even smaller quantities of por-

- C’S

FIG. dication

2. MCD (-) and absorption (-- -) spectra in 0.1 N trifluoracetic acid in benzene.

of uroyorphyrin

octamcthyl

ester

88

KALMAN

ET

AL.

phyrins can be detected by fluorescence spectroscopy; however, the proclivity of this technique to interference from impurities which are tither fluorescent, themselves or else are effective quenchers of porphyrin fluorescence is well known. Consequently, MCD is again the method of choice. Another out’standing feature of this analytical method for porphyrins arises from the disparity in the molecular ellipticities found for the porphyrin dications (16) as compared to ot#her organic chromophores, excepting, of course, the Ddh metal complexes, which have been measured to date (12). For example, the peak-to-peak amplitude of the MCD bands in the Soret region of uroporphyrin octamethyl ester dication, Fig. 2, is 126 deg cm? dimole-’ G-l. For comparison, we take the purine rhromophore, or more specifically the 6-amino derivative, adenine. as an example of a compound whose derivatives are commonly encountered in biological systems. The difference in the peak-to-trough ellipticities measured for the two oppositely signed B terms at about 260 nm in the MCD spectrum of adenine is 1.2 deg cm” dimole-’ G-’ (19)-a onehundred-fold difference. Another example is the nonheme iron protein, xanthine oxidase (20). This protein has rather strong absorption around 400 nm but surprisingly small MCD in this region. As a final example. flavin-adenine dinucleotide (FAD) shows a negatively signed MCD band close to 400 nm but the molecular ellipticity reported (20) for this compound, 0.32 deg cm2 dimole-l G-’ is roughly 400 times smaller as compared to the dication derivative of uroporphyrin octamethyl ester (Fig. 2). Consequently, it is not unreasonable to suppose that there will bc relatively little interference from other absorbing compounds that may be present in urine. This will also be the case for porphyrinogens (vi& infral and for products of heme catabolism such as bilirubin, etc., since AlCD spectra such as those shown in Figs. 1 and 2 are exhibited only by cylized, fully conjugated tetrapyrroles. The apparent absence of troublesome interferences is, of course, a boon to any analytical procedure but in the present case-the determination of porphyrins by MCD-the consequences are especially far reaching. In particular, it means that the usual tedious purification steps should be required only on rare occasions. Thus, sample handling is reduced to the addition of HCl to form the dication whereas methods based on absorption or fluorescence spectroscopy require solvent extractions and, frequently, chromatography. As a result the precision and accuracies attainable using the MCD procedure should be quite high. Furthermore, these factors, coupled with the relatively large signals involved, indicate that measurements can be made with sufficient rapidity that its application as a viable screening method becomes not only feasible but practicable as well. An example of the MCD curve of a urine specimen from an adult

DETERMINATION

OF

PORPHBRIKS

BY

MCD

89

is shown in Fig. 3. The measurement of this curve was completed in less than 10 min. The capability of obtaining data efficiently and rapidly further enhances the precision and accuracy of the method since the samples are exposed to room light for much shorter times, thereby mitigating the opportunity for photochemical reaction. It is also important to note that the main features observed in the MCD spectra of the dication derivatives of copro- and uroporphyrin arc typical of the majority of porphyrin dications measured to date (16). In part.icular, the sign pattern of the lobes of the il terms associated with the Soret and Qo-li absorption bands is negative then positive on going to shorter wavelengths; the cross-over points of these B terms are in the near vicinity of 410 and 590 nm, respectively; and the intensity of the B term in the Soret region is approximately 6 times that of the A term correlating with the QO-, transition. Although porphyrins having substituents which interact strongly with the ring r-elect’ron system may exhibit, quit’e different XICD curves, e.g., chlorins and rhodoporphyrin derivatives (16)) organic systems (save for metallo porphyrins) having MCD spectra similar to those shown in Figs. 1 and 2 are not known, at least to the authors. Consequently, the observation of these basic features in the NC’D of an acidified clinical specimen can be taken as definitive evidence for the presence of porphyrin dications.3 Indeed, the finding of an S-shaped band at about 410 nm, in the course of the restricted wavelength scan shown in Fig. 3 can be viewed with nearly the same level of confidcncc. By comparison, absorption and fluorimetric measurements on most clinical samples are likely to be, to a large extent, equivocal because of impurities. The experimental procedure. Crine samples were protected from room light and allowed to come to room temperature. A convenient aliquot, usually 9 ml, was removed from the specimen cont’ainer and made 0.2 iV in HC’l. Precipitates were removed by centrifugation when necessary. A particular sample cell, having a path length of 1 cm and requiring 1 ml of sample was used for each measurement. The outside diameter of this Teflon cell was 2.5 cm which permitted it to be placed in the room temperature bore of a superconducting magnet built (21) by Lockheed Palo Alto Research Laboratories (Model OSCM-103’1. The magnetic field strength used was 49.5 kG. Reproducible centering of the sample cell well within the region of 0.570 field homogeneity was assured by means of an aluminum center stop. Loading and removal of the cell from the magnet, bore was accomplished without removal or repositioning of the magnet ’ Although many metal complexes of porphyrins these derivatives are not cspectrd to hr lwsmt qunntitir3.

show in urinnry

roughly similar features, s:tmples in appreciable

90

FIG. 3. MCD adult male. The The calibration path length is 1 0.00002/cm. Each

KALMAX

ET

AL.

curve of the total urinary porphyrins in a sample from a normal total porphyrin content is determined from the calibration ticks. standard was uroporphyrin octamethyl rster in dil. HCl. The cell cm; the urine is 0.2 N in HCl; and the sensitivity setting in b A = division is equal to 0.01 +- 0.005 p&ml “total porphyrin.”

in the circular dichroism spectrometer. The circular dichrometer urcd for these measurements is formally designated as the Durrum JASCO-Model ORD/UV/CD-5, although modifications made by Durrum Instrument Company have enabled measurements to be made using instrument sensitivity settings of a A = 0.00002Jcm and at the signal-to-noise level illustrated in Fig. 3. The scanning speed and the instrument time constant used were governed by the optical density of the sample which, howcvcr, was not allowed to exceed 2. Highly colored samples were diluted as required. The scanning speed and time-constant settings for normal samples, e.g., Fig. 3, were 8 nm/min and 1 set, respectively. The urinary porphyrin content is determined by comparison of the peak-to-trough amplitude of the Soret’ A term with calibration values. The reference standard for this preliminary study was uroporphyrin octamethyl ester (Mann Laboratories) although for an in-depth investigation, coproporphyrin would be t,he preferred standard. Other heme precursors can be routinely determined with on1.v slight modifications of the procedure just outlined being required. For example, Gibson and Goldberg (18) found porphobilinogen (PBG 1 excretion significantly increased in the kidneys of rabbits when lead intoxication was experimentally induced. The standard method for quantit’ative de-

DETERMIFATION

OF

PORPHYRIKS

BY

MCD

91

termination of PBG is a calorimetric test’ using Ehrlich’s reagent which, however, is not specific for PBG (9). A reaction that is appropriate for MCD is carried out by heating urine, made 0.034 N in HCl, in complete darkness for 30 min (221. The products formed are the uroporphyrin isomers in statistical distribution. Porphyrinogens may constitute a large percentage of the total porphyrins found in urine (18). These substances have incompletely oxidized rings and can be readily transformed into the corresponding porphyrin by oxidation with aqueous iodine (22 1. Urinnry Porphyrins by Abso~rption Specfrophotornetry A selected set of samples of 100 ml each from a 24-hr collection were sent to Bio-Science Laboratories, Van Nuys, California, for analysis. Their method is briefly described in a recent catalog (23). “Coproporphyrin is extracted with ethyl acetate. -4fter conversion of any coproporphyrinogen to coproporphyrin, the coproporphyrin is re-extracted into dilute HCl. Uroporphyrin is extracted with n-butanol, then re-extracted into HCl. Both porphyrins are then quantitated by spectrophotometric csamination of the HCl extracts at the Soret peak and comparison made with known porphyrin absorptivities.” Lead in t’rinar

y Samples

The lead content of urinary sampleswas determined on a Perkin-Elmer Atomic Absorption Spectrometer, Model 403, using the 2203 A line. The sensitivity of the method is 2 pg per 100 ml. The method is adapted from that of Berman (24). A 30-ml sample of urine is acidified to pH 2.2-2.8 by addition of trichloroacetic acid (5%). It is then placed into a flask to which are added 1 ml of ammonium pyrrolidine dithiocarbamate (1%) and 5 ml of methylisobutyl ketone (MIBK), previously saturated with water. The mixture is shaken for 2 min by hand, and then separated by centrifugation. The aqueous layer is removed. The lead is determined in the organic phase against standards prepared from lead chloride by the same extraction procedure. The zero absorption signal is set while burning water-saturated MIBK. About 4 ml of organic phase is required for each determination. Reagent grade chemicals are used and glassware is washed with hot nitric acid. The dithiocarbamate reagent must be made up daily.

The Occupational Health Division of the California Public Health Department performed these analyses using the method of Davis and Andelman (25 1. Two ion-exchange resins are used in this procedure. First, urine is pa~cd through an anion-cxphange resin, AG l-98, 200-400 mesh, in the acetate form. Porphobilinogen is held on the column. Sext, the

92

KALMAN

ET

AL.

effluent from this column is placed on a cationic-exchange resin, 40 WY4, 200-400 mesh, in the hydrogen form. This column retains ALA hut allows urea to pass freely. After washing with water until the eluant is free of urea, ALA is then eluted directly by washing with 1 M sodium acetate. ALA is then determined by calorimetric analysis. The eluant containing ALA is diluted with an acetate buffer, heated in a boiling water bath for 10 min, cooled to room temperature and an aliquot is mixed with a modified Ehrlich’s reagent. This reagent contains 2 g p-dimethylaminobenzaldehyde and 0.35 g mercuric chloride in 84 ml of glacial acetic acid and 20 ml of 70% perchloric acid. The final volume is made up by dilution to 110 ml with distilled water. The reagent develops a red color in the presence of ALA. Quantitation comes from measuring the absorbance of the solution at 553 nm. RESULTS

The analytical data tions between lead in that these correlations the persons employed serious lead poisoning; tests to indicate some for two of the workers porphyrins determined

Relation

Between

DISCUSSIOK

collected in Table 1 shows that there are correlaurine, porphyrin in urine, and ALA in urine but are very rough indeed. It is evident that none of by the tree-care company exhibited symptoms of however there is sufficient agreement among the degree of metabolic disturbance due to lead intake (No. 6 and 12). The analytical data for urinary by MCD and by a private clinical laboratory

Lead,

TABLE Porphyrins,

Subject

6 12 4 22 37 38 8

AND

1 and ALA

in the Urine

Porphyrin* h/ml) -d

20.0 -d 6.0 10.0 10.0 5.0

11 2

-d

16

10.0

2

0

of Subjects b-ALA (mg/lOOmlF

,410 ,343 ,125 OUR ,004 ,080 ,076 ,069 ,068 ,031

a Normal values for lead in urine are about 8 pg/lOO ml or below (7). b Normal values for porphyrins in urine by MCD are about 0.06GO.10 normal value from fluorimet,ric analysis (10) is about 0.06 pg,‘ml. c The normal value for AL.4 in urine is about) 0.5 mg/lOO ml. d Analysis not carried out.

.47 .47 31 .1[3 .1n .16 .48 .32 .49 45

pg/ml.

The

DETERMINATION

OF

PORPHYRINS

BY

93

3ICD

(&o-Science) are compared in Table 2. It is apparent that the two methods give comparable results. Moderately elevated urinary porphyrin levels were found for only a few members of the group. Since the urinary lead level was measured for only one of these persons, it is interest,ing that this person also 4~0~s elevated urinary porphyrin and ALA levels. The high urinary porphyrin level determined by MCD for the second worker, So. 6, receives support from the finding of an elevated level of ALA. Neither of these workers Comparison

MCD Sample No. 6 12 1 9 7 4 19 32 3 5 22 37 35 21 3x 36 8 11 2 20 1s 15 17 10 14 16 13 31 33 34 4 Bio-Sciences

Between Attalvsis

TABLE 2 MCI) Method for Total Polphyritts by Conventional RIethods

attd

method Porphyrins (bdml) -___

Conventional -_____Copro ..________

,410 ,343 . l.i2 .1.52 .181 .125 .I20 112 : 106 lo:! O!M 094 ,093 .085 .080 ,077 ,076 069 .068 054 .050 ,042 ,040 ,040 03x .0:51 ,030 ,024 ,024 0““su Laboratories,

,045 ,061

1: 1’0 ~__ .009 03 1

~~__~~

-Total ~~--~~~ .054 00”.e

,037

.o”l

0%

,014 .021

.0x .016

.04 ,037

,001 .01n

.OlO ,007

,011 ,020

ow

004

ooti

.005

01s

.01:3 see Ref.

method” .~___

23.

_

94

KALMAN

ET

AL.

exhibited overt clinical signs or symptoms of lead poisoning and, in fact, urinary samples taken a few months after the spraying season showed a return to normal values for lead and porphyrins. Lack of evidence of accumulation of lead to any serious ext’ent was not unexpected in this group because of the short period during which lead arsenate is used each year. There is concern, however, over the mode of its intake, namely through respiration of the “fog-like” spray. It is known that as much as 307% of inhaled lead may be absorbed, depending 011 part’icle size. The work reported in this communication indicates that MCD spectroscopy is a viable analytical method for the detection of porphyrins in urine. Furthermore, it is evident that this method is part’icularly suited for definitive investigat,ions where there is need of a method that is rapid and which provides analyCc,al results of high qualit’y. ACKNOWLEDGMENTS We want to thank Dr. P. I,. Wolf, Director of the Clinical Laboratory, Stanford Medical Center, Stanford, California, for making certain of his facilities available to us. The partial financial support provided by the Stanford Center for Materials Research is gratefully acknowledged. REFERENCES 1. NICAXDER OF COLOPHON (1953) The Poems and Poetical Fragments (A. S. F. Gow and A. F. Scholfield, ed. and trans.). p, 99, Cambridge University Press, Cambridge. 2. CHISHOLM, J. J., JR. (1971) Sci. Amer. 224(2) 15. 3. HERNBERG. S. AND NIKKANEM. J. (1970) Lnncet Jan. 10. 63. 4. MILLER, J. A.. BATTISTINI, V., CUMMINO. R. 1,. C., CARSWELL. F. AND GOLDBERG. A. (1970) Lnncet Oct. 3, 695. 5. BRYCE-SMITH, D. (1971) Biologist 18, 52. 6. HARDY. H. I,., CHAMBERLIN. R. I., MALOOF, C. C.. T~ornsx-. G. W.. JR. AND HOWELL. M. C. (1971) Cl&. Pharmacol. l’her. 12, 982. 7. HANOK, A. (1969) Manual for Laboratory Clinical Chemistry, p. 240. Geron-X Publishers. Los Altos, Cnlif. 8. “Analytical Methods for Atomic Absorption Spectrometry.” Pcrkin-Elmer, Norwalk, Conn. (Based upon Reference 24). 9. FERNANDEZ, A. A. ASD JACOBS. 5. L. (1970) Stand. Methods Clin. Chem. 6, 57. 10. MARTISEZ, C. A. AND MILLS. G. C. (1971) Clin. Chem. 17, 199. 11. Data sheet supplir>tl by the Whittnkcr Corporation. Space Sciences Division, Waltham, Mass. 12. DJERASSI. C.. BUSNENHI:RO. E. .4ND ELDER, D. 1,. (1971) Plrre crntl App/. Chem. 25, 57. 13. WEISS. G.. I~OBAYASHI. H. ASD GOUTERMAN. M. (1965) J. Mol. Spectrosc. 16, 415. 14. SUNDROM. M. (1968) Actrr Chem. Scnnrl. 22, 1317. 15. MLLEY. M.. FEHER. G. .~ND MA~VZISRALI,, D. (1968) J. ilfol. Spctmw. 26, 320. 16. B.w~H. G.. LIXDI~R. R. E.. BL-SNENBERO, E. AND DJERASST. C. Ann. N. I*. Acnd. Sci., (acvrpted for publirntion 1972).

DETERMINATIOS

17.

PERNIN,

M.

H.,

OF

GOI-TERMAN,

M.

PORPHX-RISS

ANI)

PERRIX.

BY

C.

1,.

9.5

MCD

(1969)

J. (‘kern.

Whys.

63. Ii:. .ISD

D.JJ:R.ASSI.

c’.

50,

4137. 18. GIBSON, 19. VnmmH.

8. L. M. AND W.. BARTH.

J. Amer.

23.

C’hem.

Eio-Scipncc T&s,”

Sot.

GOLDBERG. A. G.. RECORDS,

Ed..

38,

(1968)

90, 6163.

Ihoratorks 9th

(1970) C/k. Sci. R., ~~LTIW:NIS:R~:.

1,. 141.

Catnlog

(1971)

“Slwidiz~vl

Di:qwostic~

T.:dmmtor>