efficiency is expressed as count rate of combusted sample divided by count rate of direct'ly counted standard sample. The quenching in the two cases was measured by addition of known amounts of H3 and C14 toluene standards. The counting efficiency of H3 and C14 measured after combustion of reference standards with variable amounts of biological material i s shown in Table I. The efficiency, as defined above, for both isotopes was found to be consistently close to 90%. Interestingly enough, the efficiency remains the same with or without biological material in the range of 0.2 to 2.5 mg. Further, the efficiency of H 3 and C14 in the presence of different tissues was also found to be close to 90% (Table 11). Table 111 presents the relative counting efficiency of H 3 and S35 for labeled biological materials between two met.hods. As may be seen, the in-vial combustion method gives about 1215% higher efficiency for H 3 than with direct counting, even with samples of bile and urine as small as 10 11. Similarly, the counting efficiency (count rate combusted samplejcount rate directly counted sample) for was l2-17% higher than that of direct counting. The reproducibility of the method for single compounds of all three
Table 111.
Comparison of Counting Efficiency for H 3 and S35 between the In-Vial Combustion and Direct-Counting Methods
Substance ~
3
-
In-vial combustion Direct-counting Amount, Count rate, Efficiency, Count rate, Efficiency, PI. c.p.m." 7c c.p.m." %d 10
m
5599
90. Ob
4832
77.7
8858 75.3 10 10588 90.0 Ha-urine 51.4 68. 8c 4746 10 6355 5 3 5 -NatSOa 68.8 8549 56.5 10 10414 S3~-mucopolysaccharide a Based on average of three separate determinations. * Based on counting efficien-y for H3 of about 90% measured after combustion of Hastandard. c Calculated on the basis of known amount of activity (9240 d.p.m. in 10 rl. of Sa6standard Sp. Act. 0.3 pc./pg.). d In relation to the efficiency of the in-vial combustion method.
isotopes is demonstrated by the linearity of the calibration curves shown in Figure 3. ACKNOWLEDGMENT
The author thanks Joan Cahill and Nick Ingoglia for their skillful technical assistance and B. S. Danes for supplying the S35-mucopolysaccharide. LITERATURE CITED
( 1 ) Baggett,, B., Presson, T. L., Presson, J. B., Coffey, J. C., Anal. Biochem. 10, 367 (1965).
(2) Conway, W. D., Grance, A. J., Ibid., 9,487 (1964). ( 3 ) Davidson, J. D., Feigelson, P., Intern. J. A p p l . Radiation Isotopes 2, 1 (1957). (4) Dobbs, H. E., ANAL.CHEM.35, 783 (1963). (5) Jacobson, 11. I., Gupta. G. N., Fernandez, C., Hennix, S., Jensen, E. V., Arch. Biochem. Biophys. 86,89 (1960). (6) Kelly, R. G., Peets, E. A , , Gordon, S., Buyske, A,, Anal. Biochem. 2, 267 (1961). ( 7 ) Oliverio, V. T., Denham, C., Davidson, J. D., Ibid., 4, 188 (1962). RECEIVEDfor review April 4, 1966. Accepted July 15, 1966. Work su ported by Health Research Council of t i e City of New York, Grant No. U-1501.
Spectrophotometric Determination of Carnosine and Anserine in Muscle CHARLES J. PARKER, Jr. Wayne State University School of Medicine, Detroit, Mich.
b The reaction of carnosine with diazotized p-bromoaniline to give a red color and the reaction of carnosine and anserine with fluorodinitrobenzene to give yellow derivatives is the basis of a method for the spectrophotometric determination of these compounds in rabbit skeletal muscle. The method can be applied directly to acid extracts of muscle, and is suited for rapid routine analyses of a large number of samples. The results are in excellent agreement with those obtained by the amino acid analyzer.
C
(B-alanylhistidine) and anserine (B-atanyl-l-methylhistidine) are unique constitutents of vertebrate skeletal muscle. Although there are some exceptions ( 1 2 ) , both coinpounds are usually found together in varying proportions, depending upon the species. Nothing is known of their ARNOSINE
function. One of the difficulties in the study of the metabolism of carnosine and anserine has been the lack of specific procedures for their determination. Carnosine can be determined by the diazo procedure of MacPherson (6) based on the Pauli reaction (7) for unsubstituted imidazoles. Since anserine does not react with diazotized aromatic amines, it does not interfere in the determination of carnosine. However, a number of compounds present in muscle react with diazonium salts to give colored derivatives and to date there has been no evaluation of the suitability of this method for the determination of carnosine in crude muscle extracts. The determination of anserine has presented great difficulty, as this compound shows no specific color reactions suitable for direct analysis. A number of indirect procedures have been used for its assay (11). To date, however, the only
reliable method for its determination in biological material is ion exchange chromatography (I, 5 , 9). Chromatography is time-consuming, however, and is not suitable for routine analysis of a large number of samples. The work described below is an evaluation of the MacPherson method for the direct assay of carnosine in crude extracts of rabbit muscle and of the possibility of determining total carnosine and anserine in such extracts directly with the nonspecific amine reagent 2,4 - dinitro - 1 - fluorobenzene (FDNB). All studies were carried out with rabbit tissue. The results presented indicate that the methods are applicable to tissue extracts. Although there are compounds present in muscle extracts that react with the two color reagents used, they are not present in sufficient amounts to cause significant interference. VOL. 38, NO. 10, SEPTEMBER 1966
1359
EXPERIMENTAL
Apparatus. Absorption curves were made on a Beckman D B spectrophotometer coupled to a Sargent SRL recorder; light path 1 cm. All other absorbance measurements were made on a Coleman Universal spectrophotometer. Amino acid analysis was carried out on a Technicon amino acid analyzer. Diazotized p-Bromoaniline. Diazotized p-bromoaniline is prepared according to the procedure of Koessler and Hanke (3) for the diazotization of sulfanilic acid.
2,4-Dinitro-l-fluorobenzene Solution. Dinitrofluorobenzene (0.4 ml.) is dissolved in 50 ml. of 95% ethanol. Reagents. Carnosine, anserine, creatine, and creatine phosphate were obtained from Calbiochem, Los Angeles, Calif. Carnosine and anserine were used as standards without further purification. Adenine nucleotides were obtained from P-L Biochemicals, Milwaukee, Wis. All other chemicals were reagent grade. Determination of Carnosine. To 1.0 ml. of the solution to be analyzed, 1.0 ml. of 0.04M Versene, 1.0 ml. of
r
500
400 wave Iengt h, mp
Figure 1 . Absorption spectra of 2,4dinitrophenyl derivatives of carnosine and anserine
0.5 rnM . - _Anserine, _
Carnosine, 0.5 mM
20% NazCOs, and 2.0 ml. of diazotized p-bromoaniline soJution are added. The reaction is stopped exactly 5 minutes after the addition of diazonium salt by the addition of 2.0 ml. of 95% ethanol. The ab-
sorbance is measured against a reagent blank at 500 mp. Determination of Carnosine plus Anserine. The method is a modification of a procedure for amines described by RlcIntire el al. (4). To 1.0 ml. of the solution to be analyzed are added 0.8 ml. of 4.0% T\'aHCOJ and 0.2 rnl. of FDNB solution. The mixture is heated for 30 minutes at 55" C. At the end of this time 0.1 ml. of 10N NaOH is added to convert the unreacted FDNB to 2,4-dinitrophenol. The mixture is made acid by the addition of 1.0 ml. of 20% HC1 and extracted with 2.0 ml. of ether for 1 minute. The absorbance of the aqueous phase is measured against a reagent blank at 430 rnp. Preparation of Muscle Extracts. Muscle extracts are prepared by homogenizing fresh tissue (0.5 to 1.0 gram) in 3 volumes of 1N perchloric acid for 1 minute in a semimicroWaring Blendor. The homogenate is heated for 5 minutes a t 100" and filtered. The filtrate is neutralized and diluted to give a final dipeptide concentration between 0.05 and 0.15 mM. RESULTS AND DISCUSSION
Table 1.
Determination of Carnosine and Anserine in Rabbit Muscle" Total mosine carnosine ____ Anserine
~
~
Gastrocnemius Psoas Psoas Gastrocnemius
A
7.8 7.8 20.6 12.8 2.8 3.0 22.9 20.1 c 3.2 3.2 20.4 17.2 D 6.2 ... 18.7 12.5 Psoas D 7.6 ... 21.7 14.1 Psoas De 6.4 ... Zi .4 i5.0 0 All values mmoles er kg. of wet tissue. * Total carnosine an{ anserine minus carnosine found by diazo method. c Tissue extracted with boiling water only.
Table 11.
B
... ...
Interference of Adenine Nucleotides, Creatine, and Creatine Phosphate in Determination of Carnosine
Compound ATP
ADP
Concn., mM
5.0 5.0 10.0 10.0 2.1 2.1 4.2 4.2 8.4
AMP
Creatine
Creatine phosphate
1360
12.9 19.0 18.5
5.0 5.0 12.5 12.5 5.0 5.0 10.0 10.0 0.5 10.0 20.0 20.0
ANALYTICAL CHEMISTRY
Carnosine, mM Added Found 0,100 0.105 0.100 0.108 0.100 0.116 0.100 0.120 0.050 0.049 0.050 0.498 0.500 0.050 0.051 0.050 0.050 0,050 0.054 0.056 0.050 0.050 0.050 0.051 0.050 0.053 0.050 0.054 0.100 0.097 0.100 0.099 0,100 0.099 0.100 0.100 0.050 0.048 0.050 0.052 0.050 0.058 0.050 0.055
Compound/ carnosine 50 100 42 84 168 100 250 50 100 10 200 400 400
The color produced with carnosine by the diazo procedure follows Beer's law over the concentration range studied (0.02 to 0.2 mM carnosine). Sufficient Ni and Cu are dissolved out of the Monel metal Waring Blendor during the perchloric acid extraction of the tissue to cause serious interference in the diazo reaction. This interference is eliminated by adding Versene to the reaction mixture. The yellow colors of the 2,Cdinitrophenyl derivatives of carnosine and anserine, obtained as decribed, are linear with concentration over the range 0.0 to 0.5 mM carnosine or anserine. The data in Figure 1 show that these colors have similar absorption curves and have the same absorbance between 400 and 450 mp. Although greater sensitivity can be obtained a t 360 mp, the difference in absorbance requires the use of correction factors. In the studies described below, all measurements were made a t 430 mp. The amino acid distribution in rabbit skeletal muscle (gastrocnemius) is shown in Figure 2. Similar patterns were obtained with psoas muscles. Carnosine and anserine are the predominant amines present. Of the amino acids that react with diazotized p-bromoaniline (see discussion of interfering compounds below) only a small amount of histidine can be detected. This, along with the fact that 2,4dinitrophenyl derivatives of neutral and acidic amino acids can be extracted into ether from acid solution, suggested that carnosine and anserine could be determined by the procedures described above. The results in Table I show this
9'1Y
0.21
h)
I
-I
I
I
4
U C
1
1
12
8
m
anserine carnosine I
14
I
I
16
I
I
18
I
1
graphic procedures. The diazo and FDNB procedures were designed for use with rabbit skeletal muscle. In muscles where the carnosine content is low-e.g., fish, birds, and some reptiles (If)-and where the total carnosine and anserine content may be low, the interfering compounds discussed above could introduce error. It is advisable, therefore, that preliminary comparisons of the methods described herein with specific chromatographic procedures precede their application to muscles from species other than the rabbit.
20
hours Figure 2. extract
Amino acid composition of rabbit gastrocnemius
to be the case. There is excellent agreement between the values found by the diazo and FDNB method and those obtained by the amino acid analyzer. The results indicate that there are no compounds present in the muscles studied which cause serious interference. The variation in carnosine and anserine content between different muscles is not surprising. du Vigneaud (I I ) has described similar variations and Severin and Yudaev (8) have shown that the ratio of anserine to carnosine varies in the rabbit with age. The ages of the animals used in this study were unknown. Compounds other than carnosine and anserine normally found in muscle which react with diazotized p-bromoaniline and F D N B can be divided into two groups: those present in amounts equal to or greater than carnosine or anserine (adenine nucleotides, creatine, and creatine phosphate); and those present in trace amounts (amino acids). Although these compounds are not a problem in the muscles studied, they do impose limitations on the possible a p plication of the method to other types of tissue. Muscle extracts prepared as described under methods contain mixtures of adenosine tri-, di-, and monophosphate as well as creatine and creatine phosphate due to the hydrolysis of adenosine triphosphate and creatine phosphate-normally found in m u s c l e when the homogenates are heated to boiling in the presence of perchloric acid. Table I1 shows the analytical recovery of carnosine assayed by the diazo procedure in the presence of varying concentrations of adenine nucleotides, creatine, and creatine phosphate. Adenosine triphosphate interferes slightly when the ratio of nucleotide to carnosine exceeds 50. The interference due to adenosine di- and monophosphate is less serious, occurring only when the ratio of nucleotide to carnosine is greater than 80 and 100, respectively. Creatine
does not interfere over the concentration range studied. Creatine phosphate, however, causes some interference when the ratio of creatine phosphate to carnosine exceeds 200. The adenine nucleotide content of striatedmuscleisbetween 5 and 8 mmoles per kg. of wet tis& (8). Total creatine and creatine phosphate vanes between 20 and 30 mmoles per kg. of wet tissue (2). In those muscles where carnosine is found the amount is generally 1.0 mmole per kg. of wet tissue or greater (11). The ratio of total nucleotide plus creatine and creatine phosphate is well below that which would interfere in the analysis of carnosine. Adenine nucleotides, creatine, and creatine phosphate do not interfere with the FDNB procedure. Table I11 shows that of the amino acids which react with diazotized pbromoaniline, only histidine and tyrosine can cause serious interference if the carnosine concentration is low (about 2.0 mM or less). Arginine, phenylalanine, and tryptophan give yellow colors with the diazo reagent with absorption maxima below 450 mp. They cause no significant interference a t the concentrations found in muscle. The basic amino acids arginine, histidine, and lysine all interfere with the FDNB procedure for carnosine and anserine if the ratio of total carnosine plus anserine to total basic amino acids is less than about 7 (Table IV). In the rabbits studied, this ratio is about 29. Tallan (9) reports 33 for the rabbit and 19.5 for the cat (10). The basic amino acids present no serious problem in the determination of carnosine and anserine in the muscles of these animals. Amino acids containing hydroxyl groups interfere to a lesser extent than the basic amino acids. The determination of carnosine and anserine in muscle by the methods described is well suited for rapid analysis of a large number of samples and in this respect has a distinct advantage over the more time-consuming chromato-
ACKNOWLEDGMENT
The author is indebted to R. K. Brown who performed the amino acid determinations by amino acid analyzer, and to T. R. Needom for her able technical assistance. He is particularly
Table 111. Interference by Amino Acids in Determination of Carnosine0
Required to cause
1.0% error
in determination of Amino acid Arginine Phenylalanine TrvDtoDhan " _ -
carnosineb
1.0 mM
Found in muscle"
1.30 1.00 2.00
0.415 0.049
None d e tected Histidine 0.012 0.039 Tyrosine 0.028 0.097 Carnosine ... 7.8 Values in mmoles per kg. of wet tissue. b Calculated from molar absorbance of compounds studied. c Rabbit A, Table I. Determined by amino acid analyzer.
Table IV. Interference by Amino Acids in Determination of Carnosine plus Anserine.
Required to cause 1.0% error in determination of 10 mM
Amino acid Arginine Histidine Lysine Proline Serine Threonine
+
anserine carnosineb 0.450 0,200 0.650 1.85 1.67 2.67
Found in musclec 0.415 0,039 0.256
None detected 0.187
None detected
Carnosine ... 7.8 Anserine ... 12.8 Values in mmoles per kg. of wet tissue. Calculated from molar absorbance of compounds studied. Rabbit A, Table I. Determined by amino acid analyzer.
VOL 38, NO. 10, SEPTEMBER 1966
1361
grateful to Wilson D. Langley, State University of S e w York a t Buffalo, for his helpful discussions during the preliminary phases of this study. LITERATURE CITED
(1) Hamilton, P. B., ANAL. CHEM.35, 2055 (1963). (2) Hamoir, G., “Biochemist’s Handbook,” Cyril Long, ed., p. 666, Van Nostrand, Princeton, N. J., 1961.
(3) Koessler, K. K., Hanke, M. T., J . Biol. Chem. 39, 497 (1919). (4) McIntire, F. C., Clements, L. N.,
Sproull, M., ANAL.CHEM. 2 5 , 1757 (1953). (5) MchIanus, I. R., J. Biol. Chem. 225, 325 (1957). (6) MacPherson, H. T., Biochem. J . 36,59 (1942). (7) Pauli, H., 2. Physiol. Chem. 4 2 , 508 (1904). (8) Severin, S. E., Yudaev, I. A., Biokhimiya 16, 386 (1951).
(9) Tallan, H. H., Proc. SOC.Ezptl. Biol. Med. 89, 553 (1955). (10) Tallan, H. H., &loore, S., Stein, W. H., J. Biol. Chem. 211, 927 (1954).
(11) du S’igneaud, I’., Behrens, O., Ergeb. Physiol. Biol. Chem. Ezptl. Pharmakol. 4 1 , 917 (1939). RECEIVEDfor review April 29, 1966. Accepted June 23, 1966. Work supported by grants from the Muscular Dystrophy Associations of America and the National Institute of Arthritis and Metabolic Diseases, U.SP.H.S. (Grant A M 05311)
Spectrofluorometric Analysis of Rare Earth Chelates of Thenoyltrifluoroacetone, Benzoylacetone, and Dibenzoylmethane by Computer Spectrum Stripping Techniques ELIZABETH C. STANLEY,’ BRUCE 1. KINNEBERGI2 and LOUIS P. VARGA Department of Chemistry, Oklahoma State University, Stillwafer, Okla.
b Fluorescence studies of rare earth tetrakis chelates with the /3-diketone ligands thenolyltrifluoroacetone, benzoylacetone, and dibenzoylmethane in acetonitrile solution indicated the feasibility of a general method for rare earth analysis. By choosing a ligand having the proper chelate triplet state energy level, selective excitation of the resonance levels of the rare earth ions was observed allowing controlled elimination of some overlapping spectral features. Emission data from the single beam spectrofluorometer were corrected, normalized, machine plotted and a spectrum stripping program was found capable of identifying the components of a rare earth mixture b y systematic search and identification of the maximum peak wavelengths. The long wavelength cut-off of our spectrofluorometer limited observation of intense fluorescence to samarium, europium, and terbium chelates, but the wavelengths of possible intense line fluorescence are presented for all rare earths studied. Phosphorescence was observed for gadolinium and lutetium thenayltrinuoroacetone chelates a t
oo c.
B
of a favorable combination of energy level spacings, resonance energy level lifetimes, and shielded orbitals, sharp line fluorescence is observed for many rare earth chelate solutions. For several systems an electronic energy level population inversion is possible such that stimulated emission of light, laser action, has been observed, and ECAUSE
1362
ANALYTICAL CHEMISTRY
considerable work has gone into a search for potential liquid laser materials (5, IS, 17, If). Energy transfer processes involved in the fluorescence and phosphorescence of interesting systems such as chelates of the rare earths with benzoylacetone (l-phenyl-lJ3-butanedione) and dibenzoylmethane (1,sdiphenyl-l,3-propanedione) , were first studied extensively by Crosby, Whan, and coworkers (6, 7 , 1 2 , 2 0 )and later by others (S, 4 , 11). Ligands such as thenoyltrifluoroacetone [4,4,4-trifluoro1-(2-thienyl)-l,3-butanedione] have been shown to be especially promising energetically from the formation of strongly fluorescing tetrakis rare earth chelates (5,17) and a number of these have been prepared (I). Excitation of the 4f shell resonance levels of the lanthanide ion in a chelate “cage” proceeds by the mechanism of intramolecular energy transfer from the triplet state energy level associated with the complex as a whole (6,7,20). Since chelate triplet state levels vary according to the particular ligand used, selective excitation of the strong resonance levels of the lanthanide ions in any mixture may be observed for a series of ligands properly chosen for their triplet state levels as shown in Figure 1. If the lanthanide ion’s lowest resonance energy level is above the chelate triplet state energy level fluorescence line emission is not observed, but phosphorescence band emission due to transitions from the triplet state direct to the ground state may be observed (20). Thus the sharp fluorescence lines observed for many of the rare earths, and which are a unique property of the
particular metal ion, may be turned off or on allowing a systematic search and identification procedure compatible with computer spectrum stripping. This suggested, further, that the fluorescence method would fulfill the conditions of specificity and sensitivity necessary for the trace analysis of the rare earths. Previous analyses of the rare earths by a number of techniques have been reviewed (19,26). The determination of trace quantities has always presented difficulties and the fluorescence method of analysis for simple aquated rare earth ions lacked sensitivity (10). Fluorescence analyses in aqueous tungstate, oxalate, or tetraborate-containing solutions a t pH 9 were shown by .ilberti and llassucci ( 2 ) to be sufficiently sensitive and selective, but the nature of the absorbing-fluorescing species, likely dimers or higher aggregates] was not determined. I n the present paper the feasibility of a sensitive qualitative and quantitative determination for the rare earths by spectrofluorometric methods was studied using tetrakis chelate systems in acetonitrile. In addition, digital data processing methods were tested on a general program for the identification of the rare earths present in a complex mixture. EXPERIMENTAL
Reagents and Chemicals. Rare earth oxides and metals (99%) were obtained from the Lunex Co. and rare earth chlorides (99.9%) from 1 Present address, University of Illinios, Urbana, Ill. 2 Present address, University of Oklahoma, Norman, Okla.
