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NMR study of in vivo RIF-1 tumors
104
Biochimica et Bioph~,swa A eta. 805 (1984) 104 116 Elsevier BBA 11337
N M R S T U D Y OF IN VIVO R I F - I T U M O R S ANALYSIS O F P E R C H L O R I C ACID EXTRACTS AND I D E N T I F I C A T I O N O F IH, 3 1 p A N D 13C R E S O N A N C E S W I L L I A M T. E V A N O C H K O a, T E D T. SAKAI a,b, T H I A N C. N G a.,, N. R A M A K R I S H N A a.b, H Y U N DJU K I M c,**, R O B E R T B. Z E I D L E R c'**, V I T H A L K. G H A N T A d R. W A L L A C E B R O C K M A N e, LEWIS M. S C H I F F E R f, PAUL. G. B R A U N S C H W E I G E R f and J E R R Y D. G L I C K S O N a,b*** a Comprehensive Cancer Center and Departments of b Biochemistry, c Pharmacology, and d Microbiology, University of Alabama in Birmingham, University Station, Birmingham, A L 35294, e Biochemistry Department, Southern Research Institute, Birmingham, A L 35205 and / Department of Experimental Therapeutics, A M C Cancer Research Center, 6401 West Colfax Avenue, Lakewood, CO 80214 (U.S.A.) (Received December 12th, 1983) (Revised manuscript received May 8th, 1984)
Key words: Tumor metabolism," NMR," Perchlorate extract," Phosphate analysis
Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-I) tumors grown in mice have been analyzed by multinuclear N M R spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the a l p resonances observed in vivo to specific phosphorus-containing metaboiites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P - N M R spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. I-IPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The t H and I3C resonance assignments of the majority of metaholites present in R I F - I extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts. Introduction In vivo 31p-NMR spectra have been reported for a variety of tumors of routine [1-4], rat [5] and h u m a n [5-7] origin. These data provide a basis for * Present address: Division of Radiology, Cleveland Clinic Foundation, Cleveland, O H 44106, U.S.A. ** Present address: Department of Pharmacology, University of Missouri, Columbia, M O 65201, U.S.A. *** Present address: Department of Radiology, Johns Hopkins Hospital, Baltimore, M D 21205, U.S.A. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
the clinical utilization of this technique in the management of human cancer and for experimental studies of neoplasms; this is of special significance in view of the recent work of Bottomley et al. [8] which demonstrated that in a single instrument it was possible to obtain both N M R images and high-resolution spectra on humans. Meaningful interpretation of these spectra requires the rigorous identification of resonances with individual phosphorus atoms of specific metabolites.
105 Studies have shown that the region of the 31p-NMR spectrum, generally ascribed to sugar phosphates in previous investigations of a variety of normal tissues [9-11] and various types of isolated tumor cells [12,13], may contain other phosphomonoesters including lipid metabolites [14,15]. These considerations point to the need for unambiguous determination of the origin of phosphate resonances in the spectra of in vivo tumors. Here, we report the rigorous identification of the 31p resonances of the in vivo radiation-induced fibrosarcoma (RIF-1) murine tumor model developed by Twentyman et al. 17. We have analyzed the composition of perchloric acid extracts of in vivo tumors and cultured tumor cells by a variety of methods, including multinuclear N M R spectroscopy and several chromatographic techniques. The emphasis of this paper is on the identification of resonances, and no attempt has been made to correlate levels of metabolites with tumor size, state of necrosis or any other, physiological or histological variable. Many of the 1H and ~3C resonances in N M R spectra of perchloric acid extracts of RIF-1 tumors have been identified. The 1H assignments are particularly significant because of the recent observation of these resonances in spectra of intact tissues [16]. Our data provide a basis for the assignment of in vivo 1H-NMR spectra of this and probably other tumors. ~H-NMR is considerably more sensitive that 3~p-NMR and is likely to play a significant role in clinical applications of in vivo NMR.
Experimental Growth of RIF-1 tumors. A frozen cell suspension of RIF-1 cells was obtained from Dr. Robert F. Kallman at Stanford University and was passed in culture and in vivo according to the protocol of Twentyman et al. [17]. Solid tumors were induced by subcutaneous inoculation of 1 • 10 6 cells in the right flank of 2-3-month-old female C 3 H / H e N mice (Simonson Laboratories, Gilroy, CA). Perchloric acid extraction. Cultured tumor cells were extracted with perchloric acid as described by Munch-Petersen et al. [18]. After neutralization with potassium bicarbonate and removal of potassium perchlorate, the extract was passed through a column (1 x 8 cm) of Chelex-100 (sodium form;
Bio-Rad Laboratories, Richmond, CA). The lyophilized eluate was dissolved in 2 H 2 0 (Aldrich Chemicals, Milwaukee, WI), and the pHm (meter reading uncorrected for deuterium isotope effects) was adjusted to the apparent pH of the tumor which had been estimated from the chemical shift of the inorganic phosphate resonance in vivo [2,19]. Solid tumors were isolated by (1) surgical excision from the anesthetized living host (pentobarbital, 60 m g / k g , intraperitoneal), (2) removal following freezing of the whole animal in liquid nitrogen, or (3) freeze-clamping. Skin and hair were removed from the (frozen) tumor prior to extraction. In each case, the tumor was pulverized under liquid nitrogen using a precooled mortar and pestle. Perchloric acid (0.5 M) was added with replacement of evaporated cryogen, and the acidice was co-pulverized with the tumor tissue. The total volume of perchloric acid (in ml) equalled 5-times the weight of the tumor (in g). The tumoracid powder was thawed at 4 o C and the extraction was then performed as described above for the isolated tumor cells. A total of 21 RIF-1 tumors of various sizes ~vere extracted. All tumor extracts showed the same resonances but with varying intensities. Freeze-clamping. The animal was anesthetized as described above, and the shaved tumor was rapidly frozen by compression between two blocks of lead (110 mm x 65 mm X 15 mm) which had been precooled to liquid nitrogen temperature. The mouse was then killed by cervical dislocation, and the tumor was excised and weighed (while still cold), and extracted as described above. N M R spectra. Spectra of tumor extracts were measured by Fourier transform N M R on a Bruker WH-400 spectrometer (9.4 T). In vivo 31p-NMR spectra of tumors were measured on a Bruker CXP-200/300 spectrometer operating at 4.7 T, utilizing either a 20 mm outer diameter 3 turn surface coil probe or a 15 mm outer diameter solenoidal probe equipped with a Faraday shield [3]. The 31p chemical shifts of resonances of perchloric acid extracts are referenced to external phosphoric acid (85%) or to i n t e r n a l glycerophosphorylcholine ( + 0.49 ppm) [14]. Proton chemical shifts are referenced to the methyl hydrogen resonance of internal sodium 4,4-dimethyl-2,2,3,3-tetradeuterio-4-silapentanoate, and
106
carbon spectra to the methyl carbon resonance of an external sample of this reference standard. Two-dimensional N M R experiments were performed using the homonuclear shift-correlated (COSY) technique [20,21]. The sampling rate was optimized for m a x i m u m signal-to-noise ratio. Analysis of ribonucleotides was performed on a Waters Associates ALC 202 HPLC. Separations were made on a W h a t m a n Partisil SAX-10 qolumn employing a linear gradient of a m m o n i u m dihydrogen phosphate (5 mM, ph 2.8 to 750 mM, p H 3.7). Automated phosphate analyses were carried out on an analyzer developed by Bessman [22]. Anion-exchange chromatography employed in this analysis was conducted according to the method of Zeidler and Kim [23]. Amino-acid analyses were performed by Dr. William T. Butler, University of Alabama in Birmingham, on a Beckman aminoacid analyzer. Phosphorus compounds used in these studies were obtained from Sigma Chemicals (St. Louis, MO) or Aldrich Chemicals (Milwaukee, WI).
Results
Resonance assignments were made using a variety of methods including: (1) analysis of chemical shifts and their p H dependence, (2) homo- and selected heteronuclear decoupling, (3) doping with known compounds, and (4) two-dimensional proton N M R spectroscopy. Assignments were confirmed by H P L C analysis of nucleotide composition, automated phosphate analysis, and in the case of 1H and 13C resonances, by amino-acid analysis.
Assignment of 31p
resonances
The in vivo 31p-NMR spectrum of the RIF-1 tumor is shown in the inset to Fig. 1. This tumor was extracted after freeze-clamping. The protoncoupled 31P-NMR spectrum of this extract is shown in Fig. 1. In general, the high-field resonances were easily assigned on the basis of data in the literature and by the comparison with the chemical shifts of the
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Fig. 1 . 3 1 p - N M R spectrum (162 MHz) of the perchloric acid extract of a freeze-clamped 2.8 g RIF-1 tumor. The pH (meter reading) was 7.8. Spectral conditions employed: 16K data points 65 ° flip angle, 3.81 s recycle time, 1736 scans. The corresponding 31p spectrum (80.96 MHz) of the in vivo tumor is shown in the inset (512 scans, 3.2 s recycle time, 17 m m internal diameter shielded solenoid probe). PC, phosphorylcholine; PE, phosphorylethanolamine; PCr, phosphocreatine; DPDE, diphosphodiester; PME, phosphomonoesters; GPC, glycerophosphorylcholine; GPE, glycerophosphorylethanolamine.
107
H P L C (Table I). As expected, ATP is the most abundant triphosphate, but other nucleotides are present in significant amounts. A typical tumor had the following nucleotide (NTP) composition; 59% ATP; 23% UTP; 13% GTP; 5% CTP. We concluded that the small purine peak (on the high-field side of the NTPp triplet) arises predominantly from UTPp. The doublets at - 9 . 8 1 p p m on the low-field side of NTP~ and at - 5 . 3 9 p p m on the high-field side of NTPr originate from nucleoside diphosphates (NDP) a and fl resonances, respectively. A low level of N D P relative to N T P indicates that little if any hydrolysis of N T P has occurred during the extraction procedure. The relative amounts of N D P and N T P resonances correlate with the nucleotide composition determined by H P L C (Table I). The N A D peak (originating from both reduced and oxidized pyridine dinucleotides and their 2'phosphates) yields a complex multiplet centered at - 1 0 . 5 9 p p m on the high-field side of the NTP~ peak. Phosphocreatine yields a sharp resonance at - 2 . 4 8 ppm. The phosphocreatine peaks in the in vivo spectra are considerably broader and hence of lower amplitude than the corresponding resonances in the spectra of the perchloric acid extract. Sharp 31p resonances were observed only after passage of the extract through Chelex-100 or addition of 5% EDTA. This suggests that broadening of the resonances may result from the association of the metabolites with paramagnetic metal ions.
TABLE I HPLC A N A L Y S I S OF T H E N U C L E O S I D E DI- A N D TRIP H O S P H A T E C O M P O S I T I O N OF A RIF-1 T U M O R PERCHLORIC ACID EXTRACT Extract was prepared from a 0.6 g tumor. Analysis was performed on 0.20 ml samples, n.d., not determined due to an overlap with an artifact peak from buffer. Nucleotide
Concentration ( n m o l / g )
ATP GTP CTP UTP ADP GDP CDP UDP
1006 216 89 387 121 60 n.d. n.d.
resonances of authentic samples. These results were further substantiated (where possible) by the titration behavior of the resonance in question in response to changes in p H and comparison with authentic samples under the same conditions. The sharp resonance at 2.47 p p m in the perchloric acid extract spectrum arises from inorganic phosphate (Pi)- The intense resonances centered at - 4 . 9 6 (doublet), - 1 0 . 1 9 (doublet) and - 2 0 . 5 1 p p m (triplet) arise from the T, a and fl phosphates, respectively, of nucleoside triphosphates (NTP). Each resonance is split into a major component arising from purine nucleotides and a minor component arising from pyrimidine nucleotides. The relative contributions of these components mirror the nucleotide pool composition determined by
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108
The two small peaks observed at 0.49 and 1.03 ppm originate from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE), respectively [13-15]. The region of the spectrum to low field of Pi originates from phosphomonoesters. In other systems, this region has been assigned to sugar phosphates [9-11]. In many instances, these resonances are derived from various glycolytic intermediates [12,24,25]. In RIF-1 extracts, however, there is little or no evidence of these derivatives. Comparison with authentic samples shows little, if any, of the common hexose and triose phosphates. The most intense resonances in this spectral region emanate from phosphorylethanolanaine at 4.50 ppm and phosphorylcholine at 3.99 ppm. Because of a possible overlap with ribose 5-phosphate, phosphorylethanolamine was further assigned by selectively irradiating the OCH 2 proton resonance (3.99 ppm) (see below) and observing the collapse of the JP-H coupling (6.10 Hz) in the 31p-NMR spectrum. The presence of both phosphorylethanolamine and phosphorylcholine were confirmed by automated phosphate analysis (Fig. 2
TABLE II A U T O M A T E D P H O S P H A TE ANALYSIS OF A RIF-1 T U M O R P E R C H L O R I C A C I D EXTRACT Perchloric acid extract was prepared from a 6.0 g tumor that was frozen after surgical excision from the anesthetized mouse. Analysis was performed on a 0.2 ml sample corresponding to 0.6 g of tumor tissue. Peak
A mount ( n m o l / g )
ATP A D P + UTP Pi Phosphorylethanolamine phosphorylcholine N A D + hexose phosphate AMP Triose phosphate Fructose 1,6-bisphosphate + 2,3-diphosphoglycerate CTP UMP Phosphocreatine
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Fig. 3. 31p-NMR spectrum (162 MHz) of a perchloric acid extract of cultured R I F - I cells grown in Waymouth medium 752/1. The sample, contained in 2.0 nil, was prepared from 0.3 ml of packed cells. The pH of the sample was 6.8 and the spectrum was obtained in 3500 scans. The spectral conditions are as described in the legend to Fig. 1 for the extract spectrum. Abbreviations, same as fig 1.
109 and Table II). Under the conditions used, GPC and G P E could not be determined, since they were not retained on the anion-exchange column. The relatively low concentrations of ATP in this sample may reflect, in part, the advanced stage of growth of this tumor. In 31p spectra of RIF-1 extracts the phosphorylethanolamine peak is substantially more intense than the phosphorylcholine peak, whereas glycerophosphorylcholine appears to be more abundant than glycerophosphorylethanolamine. In extracts of other tumors (Dunn osteosarcoma [4], MOPC 104D myeloma and human colon xenograft (CX-1); unpublished results), the opposite trend appears to hold, indicating that differences may be specific for the type of tumor examined or they may reflect variations in the metabolic state of the tumors. Some RIF-1 extracts exhibited small, vestigial A M P and glucose 6-phosphate resonances, but these peaks were absent or below the noise level in spectra of most tumor extracts. The 31p spectrum of a perchloric acid extract of RIF-1 tissue culture cells is shown in Fig. 3. The in vitro t u m o r extract lacks the p h o s p h o r y lethanolamine peak that was prominent in the spectrum of the in vivo extract. The most intense phosphomonoester resonance originates from phosphorylcholine. The spectra of the perchloric acid extract of the isolated cells contained an additional unidentified resonance (tentatively assigned to AMP) to low field of the phosphorylcholine peak. Except for these differences, the 31p spectrum of the in vitro RIF-1 tumor resembles that of the in vivo tumor. The only major unassigned 31p resonance is a complex peak centered at - 1 2 . 1 3 ppm which is generally attributed to diphosphodiesters. On the basis of chemical shifts, we have ruled out the possibility that this peak arises from cytidine diphosphocholine or cytidine diphosphoethanolamine.
1H-NMR spectrum A typical ~H-NMR spectrum of the perchloric acid extract of a RIF-1 tumor in 2H20 is shown in Fig. 4. Well-resolved resonances of various amino acids, nucleotides, creatine, phosphocreatine, phosphorylethanolamine, phosphorylcholine, GPE, G P C and lactate are observed. Coupled res-
onances were identified by shift-correlated two-dimensional N M R spectroscopy (Fig. 5) [20,21]. The pH titration behavior of various resonances was instrumental in their assignment to specific species. The phosphocreatine and creatine methyl singlets near 3.0 ppm can be resolved from each other below pH = 7.8 (creatine is at higher field). The methylene 1H resonances of these species yield singlets near 4.0 ppm that can be resolved over the entire pH range. Hence, both components of the creatine pool can be monitored by 1H-NMR. The methyl peaks of phosphorylcholine, glycerophosphorylcholine and possibly choline are resolved singlets at pH > 4.8 with a chemical shift of about 3.2 ppm. At lower pH, these peaks merge. In a similar manner, the N C H 2 hydrogens of these species give rise to resolvable resonances near 3.6 ppm, and the OCH 2 hydrogens yield resolvable peaks near 4.2 ppm at pH > 5. A complex multiplet from the glycerol hydrogens of GPC occurs near 3.9 ppm. The most intense 1~ resonances of the tumor extract originate from taurine (2-aminoethanesulfonic acid). The presence of this compound as well as of other amino acids in tumor extracts was verified by amino-acid analysis. The region of the spectrum near 3.2 ppm is very complex as a result of overlap of resonances emanating from the taurine SCH2, phosphorylethanolamine N C H 2 and the phosphorylcholine and glycerophosphorylcholine N C H 3 hydrogens. The lactate and threonine CH 3 peaks overlap near neutral pH but are resolved in acid or alkaline solution. The low-field portion of the 1H spectrum (Fig. 4b) is less complex than its high-field counterpart and shows resonances derived primarily from adenine derivatives and some uracil- and cytosine-containing compounds. Also present were resonances of low intensity arising from phenylalanine, tyrosine and histidine or their derivatives. It should be emphasized that this portion of the spectrum has been amplified 16-fold relative to the high-field portion, and that the species giving rise to low-field resonances are present at significantly lower concentrations than those producing the more intense high-field resonances. Although qualitatively similar to the extract of the in vivo tumor, the extract of RIF-1 tissue culture cells shows some quantitative differences.
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Fig. 4. The 1H-NMR spectrum (400 MHz) of the perchloric acid extract prepared from a 7.8 g RIF-1 tumor showing (a) the high-field and (b) the low-field regions. The amplitude of the peaks in (b) is 16-times that of the peaks in (a). The p H of the sample was 6.8. Spectral conditions employed: 16K data points, 90 o flip angle, 3.05 s recycle time, 300 scans, no solvent suppression.
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Fig. 6. Portion of a 13C-NMR spectrum (100 MHz) of the RIF-1 tumor extract described in Fig. 5. The pH of the extract was 6.8. Conditions: 90 o flip angle; 3.2 s recycle time; 10000 scans. TSPext, external reference, 4,4-dimethyl-2,2,3,3-tetradeutero-4-silapentanoate.
The major difference is the lower concentrations of creatine, phosphocreatine and taurine. Several 1H resonances remain unidentified. At pH 9.1, two relatively intense coupled triplets are resolved at 3.07 and 3.42 ppm which arise from an unstable, unsymmetrically 1,2-substituted ethane
T A B L E III SELECTED C H E M I C A L SHIFTS F O R 13C R E S O N A N C E S IN A RIF-1 E X T R A C T (pH 6.8) Compound
Chemical shift (ppm)
Taurine Lactate phosphorylethanolamine phosphorylcholine Phosphocreatine Creatine Glycine Alanine Glutamic acid
38.18, 50.23 (CH2-N , CH2-S ) 22.86, 71.19 (CH3, CH) 43.37, 62.93 (CH2-N, CH2-O ) 56.73 ((CH3)3) 39.59, 60.76 (CH 3, CH2) 39.79, 56.60 (CH3, CH2) 44.24 (CH2) 18.92, 53.27 (CH 3, CH) 29.65, 36.19, 57.32 (CH2-CH, CH 2-CO, CH-CO)
derivative (i.e., observed only in flesh extracts). We have confirmed that these peaks do not originate from 2-aminoethanethiol, isethionic acid or hypotaurine.
13C_NMR The 13C-NMR spectrum of an extract from a single very large (7.8 g) tumor was obtained in an overnight experiment (Fig. 6, Table III). As in the 1H-NMR spectrum, the most intense resonances (at 38.18 and 50.23 ppm) originate from taurine. Resonances of other amino acids - alanine, glycine and glutamic acid - are observed as are resonances originating from lactate, phosphorylethanolamine, phosphorylcholine, creatine and phosphocreatine. A few resonances remain to be identified, including a fairly intense peak at approx. 163 ppm (not shown). There was a great deal of variability in the samples obtained from different tumors. Variables such as tumor size, degree of necrosis, hypoxic cell fraction, etc., all have profound effects on the metabolic state of tumors. Since the major objec-
113 tive of this paper was to identify metabolite resonances, no attempt was made to grade the tumors used in this study by size or time post-implantation, or to systematically investigate these variations. Discussion
Several N M R studies have been directed toward the identification of phosphorus metabolites in tumor cells in culture [12-15], including studies by Evans and Kaplan [12] who have extended such work to extracts of some solid human tumor xenografts in athymic mice. The 31p spectra of these intact, dispersed tumor cells all exhibit Pi, N T P and N D P resonances. However, considerable differences were observed in the phosphomonoester and diester resonances, and in the composition of the nucleotide pool. Thus, extracts of H e L a cells [12] like those of RIF-1 tumors exhibited two sets of resonances corresponding to purine and pyrimidine nucleotides, whereas extracts of Ehrlich ascites cells [14] exhibited only the purine nucleotide resonances. In the case of RIF-1 tumors, A T P was the predominant high-energy phosphate in the cell (Tables I and II); however the presence of other nucleotides demonstrated that, in general, care must be taken in attributing N T P and N D P resonances exclusively to adenine derivatives. Some ambiguity has existed concerning the identity of the phosphomonoester resonances. Evans and Kaplan [12] did not identify these resonances in their study of solid human tumor xenografts in athymic mice. In spectra of in vivo tissues these resonances have generally been assigned to sugar phosphates [9-11]. In instances where resonances of sugar phosphates have been observed, changes in these peaks have been attributed to anaerobic glycolysis [25]. However, N a v o n et al. [15] reported that virus-transformed lymphoid cell lines exhibited 31p spectra containing mostly phosphorylethanolamine and phosphorylcholine in the sugar phosphate region. We have found that for in vivo RIF-1 tumors, the phosphomonoester resonances, like those originating f r o m cultured virus-transformed lymphoid cells [15], arise almost exclusively from phosphorylethanolamine and phosphorylcholine, and glycolytic intermediates are usually below the level of N M R detection. These observations, to-
gether with similar findings on two other tumors (Dunn osteosarcoma and M O P C 104E myeloma) that we have examined, suggest that processes other than glycolysis are responsible for changes in the intensity of the phosphomonoester resonances of at least some in vivo tumors, although glycolysis does occur in these tumors as is demonstrated by the high level of lactic acid (Fig. 4a) [26]. We considered the possibility that such glycolytic intermediates may have been rapidly depleted by the trauma associated with isolation of the tumor. However, the small differences between spectra of tumors extracted after excision from the in vivo host, from the frozen host and from the freeze-clampled tumor argue against this possibility. Agreement between the chemical shifts of these resonance in spectra of the in vivo tumor and of tumor extracts further supports this conclusion. In this regard, RIF-1 tumors appear to be comparatively stable in their maintenance of high levels of phosphorus metabolites. Ribose 5-phosphate has been reported as a major metabohte in 31p-NMR spectra from guinea-pig brain extracts [27]. Our studies of this compound rule it out as a possible 31p-NMR-detectable metabolite in the RIF-1 tumor. It should be noted, however, that the chemical shifts and p H titration behavior of ribose 5-phosphate and phosphorylethanolamine are strikingly similar. We have found that for authentic samples, their chemical shifts coincide at p H greater than 7.7 and less than 4.6. They can easily be distinguished, however, by specific heteronuclear 1H decoupling experiments, which have been performed in our study but have not been reported in the study of guinea-pig brain extracts. The in vitro tumor cells did not exhibit either a ribose 5-phosphate or a phosphorylethanolamine 31p resonance, but did show a phosphorylcholine peak. The presence of choline but not ethanolamine in the growth medium could explain the presence of phosphorylcholine but not phosphorylethanolamine in the in vitro cell preparation. There is some controversy about the origin of the phosphorylcholine, phosphorylethanolamine, G P C and G P E resonances in spectra of various intact tissues [15,28-30]. Decomposition of phospholipids in regions of tumor necrosis could generate some or all of these metabolites. Alternatively, these molecules may accumulate in regions of rapid
114 tumor growth to meet the demands for biosynthesis of phospholipids. Both of these processes are probably occurring in the in vivo tumor, but it is important to determine their relative contributions to elevated levels of these metabolites. The fact that these resonances increase with increasing tumor size (and hence with decreasing growth fraction of the tumor) suggests that the catabolic mechanism is playing the predominant role in generating these metabolites. If this proves to be the case, then levels of these derivatives may serve as indices of the' extent of tumor necrosis. There has been also considerable controversy whether tumor cells produce sufficient phosphocreatine for detection by 3~p-NMR or whether phosphocreatine resonances originate from normal cells infiltrating or adjacent to the tumor. Spectra of a variety of cells in culture [14,15] and of a Walker sarcoma implanted in a rat [5] did n o t exhibit a phosphocreatine peak. However, spectra both of other subcutaneously implanted murine tumors [1-4] and of other tumor cells in culture [12,32] demonstrated the presence of phosphocreatine. The fact that phosphocreatine was detected by some investigators and not by others, in some cases for the same in vitro cell lines (either HeLa or Ehrlich ascites), suggests that environmental factors may be responsible for these differing results. In vivo studies of brains [33] and hearts [34] clearly demonstrate that even mild hypoxia is sufficient to convert phosphocreatine to creatine. At the high cell densities employed in the in vitro studies (approx. 1.108 cells/ml), some degree of hypoxia may be expected even if gases are bubbled through the cell suspension. Other effects such as accumulation of various toxic metabolites may also affect the phosphocreatine/creatine ratio. The recent study of Agris and Campbell [35] reports the presence of phosphocreatine in the aH - N M R spectrum of intact Friend leukemia cells and of their perchloric acid extracts. Since the chemical shift differences of phosphocreatine and creatine are small and sensitive to pH, it is not clear how these authors ruled out creatine as the resonance observed. This possibility is suggested by the reported absence of phosphocreatine in 3~p-NMR spectra of these cells [15]. An alternative explanation that phosphocreatine originates only from normal tissues either
proximal to or embedded in the tumor is untenable (at least in the case of the RIF-1 tumor), in view of the observation that perchloric acid extracts from pure cultures of RIF-1 cells (Fig. 3) exhibit a phosphocreatine resonance. Furthermore, r.f. field plots (with phantom samples) of the probe employed in this study clearly exclude any spectral contributions from tissues outside the tumor, a fact that was further substantiated by the absence of signals when a tumor-free mouse was placed in the N M R probe. Some blood vessels and fibrous tissues are present in the tumor and may contribute to the observed spectral resonances, but histological examination indicates that these cannot account for the majority of the spectral intensity. Lymphocyte infiltration is also known to be minimal in this nonimmunogenic tumor [17]. Whether the observed phosphocreatine results from endogenous or exogenous creatine remains to be determined.It may be that, as has been reported for Ehrlich ascites cells [32], RIF-1 tumors lack the ability to synthesize creatine, but retain the ability to phosphorylate it. In some RIF-1 extracts, an extra 31p resonance was observed approx. 0.1 ppm to low field of the phosphocreatine resonance. This signal results from coupling of the phosphorus nucleus to protons on the phosphoramide nitrogen under conditions where not all of the H 2 0 was replaced by 2H20 [36]. Because of its approx. 16-fold greater sensitivity compared to 3~P-NMR, ~H-NMR may prove to be particularly useful for clinical applications. This nucleus offers the additional advantage of directly monitoring several metabolites that cannot be readily detected by other N M R techniques. Noteworthy among these is lactate, whose elevated levels in tumors is well documented [37], and may prove useful in the diagnosis of cancer. Near neutral pH, the lactate methyl resonance overlaps with the threonine methyl peak. Overlap can be eliminated in acid solution, but since lactate is usually much more abundant than threonine, little error is introduced by ignoring this overlap. Taurine has been detected in a variety of normal and neoplastic tissues [38]. This aminosulfonic acid, which is produced from the catabolism of cysteine, exists in tissues as a free amino acid and is not known to be utilized for protein synthesis or i
115
as a source of energy. To date, the best known function of this metabolite is its conjugation with steroids in the biosynthesis of bile acids. While taurine has generally been considered nonessential, there is evidence that it is required for normal development of the retina [39]. Its role and the reason for the substantial decrease of taurine levels in the in vitro tumor remain to be determined. However, the observation of lower levels of this and other metabolites (such as creatine and phosphocreatine) in cultured cells suggests that the host plays a considerable role in determining the levels of some metabolites found in tumors. The 13C-NMR method has proven useful for analysis of metabolic pathways by monitoring the distribution of carbon atoms that have been enriched with this isotope [40]. Natural abundance 13C-NMR, which has been employed in studies of intact excised muscle [41] and in the present study of RIF-1 tumor extracts, is of limited utility because of the low sensitivity of this nucleus and the consequent requirement for long accumulation times that are impractical for in vivo applications. However, these studies demonstrate that the method is quite useful for monitoring a number of specific metabolites in the extract under conditions of minimal spectral overlap. For example, discrete ~3C resonances of lactate can be monitored without the overlap problems encountered with this metabolite in the proton N M R spectrum (see above). Resonances of taurine and glutamic acid are also better resolved in the ~3C- than in the H - N M R spectrum. These studies demonstrate the need for the unambiguous assignment of resonances from in vivo N M R spectra of tissues. The wealth of information available from multi-nuclear N M R (especially 1H) is also clearly evident. It will probably be possible to obtain at least some of this information in clinical N M R examinations of tumors and other tissues.
Acknowledgements This work was supported by USPHS Grant CA13148 (A.F. LoBuglio and J.D.G.), CA-36346 (J.D.G.), CA 33304 (J.D.G.), and Faculty Research Grant FRA-162 (J.D.G.) from the American Cancer Society. We thank Dr. Robert F. Kall-
man of Stanford University for supplying us with the RIF-1 tumor cells. N.R.K. is a Leukemia Society of America Scholar, 1982-1987.
References 1 Evanochko, W.T., Ng, T.C., Lilly, M.B., Lawson, A.J., Corbett, T.H., Durant, J.R. and Glickson, J.D. (1983) Proc. Natl. Acad. Sci. USA 80, 334-338 2 Ng, T.C., Evanochko, W.T., Hiramoto, R.N., Ghanta, V.K., Lilly, M.B., Lawson, A.J., Corbett, T.H., Durant, J.R. and Glickson, J.D. (1982) J. Magn. Resonance 49, 271-286 3 Ng, T.C., Evanochko, W.T. and Glickson, J.D. (1982) J. Magn. Resonance 49, 526-529 4 Lilly, M.B., Ng, T.C., Evanochko, W.T., Katholi, C.R., Kumar, N.G., Elgavish, G.A., Durant, J.R., Hiramoto, R.N., Ghanta, V.K. and Glickson, J.D. (1984) Cancer Res. 44, 633-638 5 Griffiths, J.R. and Iles, R.A. (1982) Biosci. Rep. 2, 719-725 6 Evanochko, W.T., Ng, T.C., Glickson, J.D., Durant, J.R. and Corbett, T.H. (1982) Biochem. Biophys. Res. Commun. 109, 1346-1352 7 Griffiths, J.R., Cady, E., Edwards, R.H.T., McCready, V.R., Wilkie, D.R. and Wiltshaw, E. (1983) Lancet i, 1435-1436 8 Bottomley, P.A., Hart, H.R., Edelstein, W.A., Schenk, J.F., Smith, L.S., Leue, W.M., Muller, O.M. and Redington, R.W. (1983) Lancet ii, 273-274 9 Balaban, R.S., Gadian, D.G. and Radda, G.K. (1981) Kidney Int. 20, 575-579 10 Burt, C.T., Pluskal. M.G. and Streter, F.A. (1982) Biochim. Biophys. Acta 721, 492-494 11 Edwards, R.H.T., Dawson, M.J., Wilkie, D.R., Gordon, R.E. and Shaw, D. (1982) Lancet, 725-731 12 Evans, F.E. and Kaplan, N.O. (1977) Proc. Natl. Acad. Sci. USA 74, 4909-4913 13 Evans, F.E. (1979) Arch. Biochem. Biophys. 193, 63-75 14 Navon, G., Ogawa, S., Shulman, R.G. and Yamane, T. (1977) Proc. Natl. Acad. Sci. USA 74, 87-91 15 Navon, G., Navon, R., Shulman, R.G. and Yamane, T. (1978) Proc. Natl. Acad. Sci. USA 75, 891-895 16 Behar, K.L., Den Hollander, J.A., Stromski, M.E., Ogino, T., Shulman, R.G., Petroff, O.A.C. and Prichard, J.W. (1983) Proc. Natl. Acad. Sci. USA 80, 4945-4948 17 Twentyman, P.R., Brown, J.M., Gray, J.W., Franko, A.J., Scoles, M.A. and Kallman, R.F. (1980) J. Natl. Cancer Inst. 64, 595-604 18 Munch-Petersen, B., Trysted, G. and Dupont, B. (1973) Exp. Cell Res. 79, 249-256 19 Roberts, J.K.M., Wade-Jardetzky, N. and Jardetzky, O. (1981) Biochemistry 20, 5389-5394 20 Bax, A. and Freeman, R. (1981) J. Magn. Resonance 44, 542-561 21 Nagayama, K., Kumar, A., Wuthrich, K. and Ernst, R.R. (1980) J. Magn. Resonance 40, 321-334 22 Bessman, S.P. (1974) Anal. Biochem. 59, 524-532 23 Zeidler, R.B. and Kim, H.D. (1982) J. Cell. Physiol. 112, 360-366
116 24 Den Hollander, J.A., Ugurbil, K., Brown T.R. and Shulman, R.G. (1981) Biochemistry 20, 5871-5880 25 Navon, G., Shulman, R.G., Yamane, T., Eccleshall, T.R., Lam, K.-B., Baronofsky, J.J. and Marmur, J. (1979) Biochemistry 18, 4487-4499 26 Wood, R. (1973) Tumor Lipids: Biochemistry and Metabolism, American Oil Chemists' Society, Champaign 27 Glonek, T., Kopp, S.J., Kot, E., Pettegrew, J.W., Harrison, W.H. and Cohen, M.M. (1982) J. Neurochem. 39, 1210-1219 28 Selkirk, J.K., Elwood, J.C. and Morris, H.P. (1971) Cancer Res. 31, 27-31 29 Koizumi, K., Tamiya-Koizumi, K., Fujii, T., Okuda, J. and Kojima, K. (1980) Cancer Res. 40, 909-913 30 Burt, C.T., Glonek, T. and Barany, M. (1976) Biochemistry 15, 4850-4853 31 Melner, M.H., Sawyer, S.T., Evanochko, W.T., Ng, T.C., Glickson, J.D. and Puett, D. (1983) Biochemistry 22, 2039-2042 32 Yushok, W.D. and Gupta, R.K. (1980) Biochem. Biophys. Res. Commun. 95, 73-81 33 Prichard, J.W., Alger, J.R., Behar, K.L., Petroff, O.A.C. and
34
35 36
37 38 39 40
41
Shulman, R.G. (1983) Proc. Natl. Acad. Sci. USA 80, 2748-2751 Gadian, D.G., Radda, G.K., Richards, R.E. and Seeley, P.J. (1979) in Biological Applications of Magnetic Resonance (Shulman, R.G., ed.), pp. 463-535, Academic Press, New York Agris, P.F. and Campbell, I.D. (1982) Science 216, 1325-1327 Kupriyanov, V.V., Steinschneider, A.Y., Ruuge, E.K., Smirnov, V.N. and Saks, V.A. (1983) Biochem. Biophys. Res. Commun. 114, 1117-1125 Warburg, O. (1931) Metabolism of Tumors, R.R. Smith Inc., New York Roberts, E. and Frankel S. (1949) Cancer Res. 9, 645-648 Hayes, K.C. (1979) Nutri. Rev. 34, 161-165 Ugurbil, K., Shulman, R.G. and Brown, T.R. (1979) in Biological Applications of Magnetic Resonance (Shulman, R.G., ed.), pp. 537-589, Academic Press, New York Doyle, D.D., Chalovich, J.M. and Barany, M. (1981) FEBS Lett. 131, 147-150
Biochimica et Bioph~,swa A eta. 805 (1984) 104 116 Elsevier BBA 11337
N M R S T U D Y OF IN VIVO R I F - I T U M O R S ANALYSIS O F P E R C H L O R I C ACID EXTRACTS AND I D E N T I F I C A T I O N O F IH, 3 1 p A N D 13C R E S O N A N C E S W I L L I A M T. E V A N O C H K O a, T E D T. SAKAI a,b, T H I A N C. N G a.,, N. R A M A K R I S H N A a.b, H Y U N DJU K I M c,**, R O B E R T B. Z E I D L E R c'**, V I T H A L K. G H A N T A d R. W A L L A C E B R O C K M A N e, LEWIS M. S C H I F F E R f, PAUL. G. B R A U N S C H W E I G E R f and J E R R Y D. G L I C K S O N a,b*** a Comprehensive Cancer Center and Departments of b Biochemistry, c Pharmacology, and d Microbiology, University of Alabama in Birmingham, University Station, Birmingham, A L 35294, e Biochemistry Department, Southern Research Institute, Birmingham, A L 35205 and / Department of Experimental Therapeutics, A M C Cancer Research Center, 6401 West Colfax Avenue, Lakewood, CO 80214 (U.S.A.) (Received December 12th, 1983) (Revised manuscript received May 8th, 1984)
Key words: Tumor metabolism," NMR," Perchlorate extract," Phosphate analysis
Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-I) tumors grown in mice have been analyzed by multinuclear N M R spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the a l p resonances observed in vivo to specific phosphorus-containing metaboiites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P - N M R spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. I-IPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The t H and I3C resonance assignments of the majority of metaholites present in R I F - I extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts. Introduction In vivo 31p-NMR spectra have been reported for a variety of tumors of routine [1-4], rat [5] and h u m a n [5-7] origin. These data provide a basis for * Present address: Division of Radiology, Cleveland Clinic Foundation, Cleveland, O H 44106, U.S.A. ** Present address: Department of Pharmacology, University of Missouri, Columbia, M O 65201, U.S.A. *** Present address: Department of Radiology, Johns Hopkins Hospital, Baltimore, M D 21205, U.S.A. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
the clinical utilization of this technique in the management of human cancer and for experimental studies of neoplasms; this is of special significance in view of the recent work of Bottomley et al. [8] which demonstrated that in a single instrument it was possible to obtain both N M R images and high-resolution spectra on humans. Meaningful interpretation of these spectra requires the rigorous identification of resonances with individual phosphorus atoms of specific metabolites.
105 Studies have shown that the region of the 31p-NMR spectrum, generally ascribed to sugar phosphates in previous investigations of a variety of normal tissues [9-11] and various types of isolated tumor cells [12,13], may contain other phosphomonoesters including lipid metabolites [14,15]. These considerations point to the need for unambiguous determination of the origin of phosphate resonances in the spectra of in vivo tumors. Here, we report the rigorous identification of the 31p resonances of the in vivo radiation-induced fibrosarcoma (RIF-1) murine tumor model developed by Twentyman et al. 17. We have analyzed the composition of perchloric acid extracts of in vivo tumors and cultured tumor cells by a variety of methods, including multinuclear N M R spectroscopy and several chromatographic techniques. The emphasis of this paper is on the identification of resonances, and no attempt has been made to correlate levels of metabolites with tumor size, state of necrosis or any other, physiological or histological variable. Many of the 1H and ~3C resonances in N M R spectra of perchloric acid extracts of RIF-1 tumors have been identified. The 1H assignments are particularly significant because of the recent observation of these resonances in spectra of intact tissues [16]. Our data provide a basis for the assignment of in vivo 1H-NMR spectra of this and probably other tumors. ~H-NMR is considerably more sensitive that 3~p-NMR and is likely to play a significant role in clinical applications of in vivo NMR.
Experimental Growth of RIF-1 tumors. A frozen cell suspension of RIF-1 cells was obtained from Dr. Robert F. Kallman at Stanford University and was passed in culture and in vivo according to the protocol of Twentyman et al. [17]. Solid tumors were induced by subcutaneous inoculation of 1 • 10 6 cells in the right flank of 2-3-month-old female C 3 H / H e N mice (Simonson Laboratories, Gilroy, CA). Perchloric acid extraction. Cultured tumor cells were extracted with perchloric acid as described by Munch-Petersen et al. [18]. After neutralization with potassium bicarbonate and removal of potassium perchlorate, the extract was passed through a column (1 x 8 cm) of Chelex-100 (sodium form;
Bio-Rad Laboratories, Richmond, CA). The lyophilized eluate was dissolved in 2 H 2 0 (Aldrich Chemicals, Milwaukee, WI), and the pHm (meter reading uncorrected for deuterium isotope effects) was adjusted to the apparent pH of the tumor which had been estimated from the chemical shift of the inorganic phosphate resonance in vivo [2,19]. Solid tumors were isolated by (1) surgical excision from the anesthetized living host (pentobarbital, 60 m g / k g , intraperitoneal), (2) removal following freezing of the whole animal in liquid nitrogen, or (3) freeze-clamping. Skin and hair were removed from the (frozen) tumor prior to extraction. In each case, the tumor was pulverized under liquid nitrogen using a precooled mortar and pestle. Perchloric acid (0.5 M) was added with replacement of evaporated cryogen, and the acidice was co-pulverized with the tumor tissue. The total volume of perchloric acid (in ml) equalled 5-times the weight of the tumor (in g). The tumoracid powder was thawed at 4 o C and the extraction was then performed as described above for the isolated tumor cells. A total of 21 RIF-1 tumors of various sizes ~vere extracted. All tumor extracts showed the same resonances but with varying intensities. Freeze-clamping. The animal was anesthetized as described above, and the shaved tumor was rapidly frozen by compression between two blocks of lead (110 mm x 65 mm X 15 mm) which had been precooled to liquid nitrogen temperature. The mouse was then killed by cervical dislocation, and the tumor was excised and weighed (while still cold), and extracted as described above. N M R spectra. Spectra of tumor extracts were measured by Fourier transform N M R on a Bruker WH-400 spectrometer (9.4 T). In vivo 31p-NMR spectra of tumors were measured on a Bruker CXP-200/300 spectrometer operating at 4.7 T, utilizing either a 20 mm outer diameter 3 turn surface coil probe or a 15 mm outer diameter solenoidal probe equipped with a Faraday shield [3]. The 31p chemical shifts of resonances of perchloric acid extracts are referenced to external phosphoric acid (85%) or to i n t e r n a l glycerophosphorylcholine ( + 0.49 ppm) [14]. Proton chemical shifts are referenced to the methyl hydrogen resonance of internal sodium 4,4-dimethyl-2,2,3,3-tetradeuterio-4-silapentanoate, and
106
carbon spectra to the methyl carbon resonance of an external sample of this reference standard. Two-dimensional N M R experiments were performed using the homonuclear shift-correlated (COSY) technique [20,21]. The sampling rate was optimized for m a x i m u m signal-to-noise ratio. Analysis of ribonucleotides was performed on a Waters Associates ALC 202 HPLC. Separations were made on a W h a t m a n Partisil SAX-10 qolumn employing a linear gradient of a m m o n i u m dihydrogen phosphate (5 mM, ph 2.8 to 750 mM, p H 3.7). Automated phosphate analyses were carried out on an analyzer developed by Bessman [22]. Anion-exchange chromatography employed in this analysis was conducted according to the method of Zeidler and Kim [23]. Amino-acid analyses were performed by Dr. William T. Butler, University of Alabama in Birmingham, on a Beckman aminoacid analyzer. Phosphorus compounds used in these studies were obtained from Sigma Chemicals (St. Louis, MO) or Aldrich Chemicals (Milwaukee, WI).
Results
Resonance assignments were made using a variety of methods including: (1) analysis of chemical shifts and their p H dependence, (2) homo- and selected heteronuclear decoupling, (3) doping with known compounds, and (4) two-dimensional proton N M R spectroscopy. Assignments were confirmed by H P L C analysis of nucleotide composition, automated phosphate analysis, and in the case of 1H and 13C resonances, by amino-acid analysis.
Assignment of 31p
resonances
The in vivo 31p-NMR spectrum of the RIF-1 tumor is shown in the inset to Fig. 1. This tumor was extracted after freeze-clamping. The protoncoupled 31P-NMR spectrum of this extract is shown in Fig. 1. In general, the high-field resonances were easily assigned on the basis of data in the literature and by the comparison with the chemical shifts of the
NrP~ P
ME
~-
PE
i
Nrg
NO~
NTP/3
ND~
~
-;
- I,0
-115
20 ppm
IPCr
NT~ NTPv
NTPa
PC
.
0
-2
-4
-6
-8
-I0
NAD
-12
-14
-16
-18
-20
ppm
Fig. 1 . 3 1 p - N M R spectrum (162 MHz) of the perchloric acid extract of a freeze-clamped 2.8 g RIF-1 tumor. The pH (meter reading) was 7.8. Spectral conditions employed: 16K data points 65 ° flip angle, 3.81 s recycle time, 1736 scans. The corresponding 31p spectrum (80.96 MHz) of the in vivo tumor is shown in the inset (512 scans, 3.2 s recycle time, 17 m m internal diameter shielded solenoid probe). PC, phosphorylcholine; PE, phosphorylethanolamine; PCr, phosphocreatine; DPDE, diphosphodiester; PME, phosphomonoesters; GPC, glycerophosphorylcholine; GPE, glycerophosphorylethanolamine.
107
H P L C (Table I). As expected, ATP is the most abundant triphosphate, but other nucleotides are present in significant amounts. A typical tumor had the following nucleotide (NTP) composition; 59% ATP; 23% UTP; 13% GTP; 5% CTP. We concluded that the small purine peak (on the high-field side of the NTPp triplet) arises predominantly from UTPp. The doublets at - 9 . 8 1 p p m on the low-field side of NTP~ and at - 5 . 3 9 p p m on the high-field side of NTPr originate from nucleoside diphosphates (NDP) a and fl resonances, respectively. A low level of N D P relative to N T P indicates that little if any hydrolysis of N T P has occurred during the extraction procedure. The relative amounts of N D P and N T P resonances correlate with the nucleotide composition determined by H P L C (Table I). The N A D peak (originating from both reduced and oxidized pyridine dinucleotides and their 2'phosphates) yields a complex multiplet centered at - 1 0 . 5 9 p p m on the high-field side of the NTP~ peak. Phosphocreatine yields a sharp resonance at - 2 . 4 8 ppm. The phosphocreatine peaks in the in vivo spectra are considerably broader and hence of lower amplitude than the corresponding resonances in the spectra of the perchloric acid extract. Sharp 31p resonances were observed only after passage of the extract through Chelex-100 or addition of 5% EDTA. This suggests that broadening of the resonances may result from the association of the metabolites with paramagnetic metal ions.
TABLE I HPLC A N A L Y S I S OF T H E N U C L E O S I D E DI- A N D TRIP H O S P H A T E C O M P O S I T I O N OF A RIF-1 T U M O R PERCHLORIC ACID EXTRACT Extract was prepared from a 0.6 g tumor. Analysis was performed on 0.20 ml samples, n.d., not determined due to an overlap with an artifact peak from buffer. Nucleotide
Concentration ( n m o l / g )
ATP GTP CTP UTP ADP GDP CDP UDP
1006 216 89 387 121 60 n.d. n.d.
resonances of authentic samples. These results were further substantiated (where possible) by the titration behavior of the resonance in question in response to changes in p H and comparison with authentic samples under the same conditions. The sharp resonance at 2.47 p p m in the perchloric acid extract spectrum arises from inorganic phosphate (Pi)- The intense resonances centered at - 4 . 9 6 (doublet), - 1 0 . 1 9 (doublet) and - 2 0 . 5 1 p p m (triplet) arise from the T, a and fl phosphates, respectively, of nucleoside triphosphates (NTP). Each resonance is split into a major component arising from purine nucleotides and a minor component arising from pyrimidine nucleotides. The relative contributions of these components mirror the nucleotide pool composition determined by
I
phosphoryl-
r2;::,-:
o
"8
pho~r= creofine
i*~orgonic
osphate
t..
l
triose
N3P°
A
1¸\
0 hours Fig. 2. Elution profile from a automated phosphate analyzer of a perchloric acid extract of an in vivo RIF-1 tumor. The analysis was performed on an aliquot of an extract prepared from a 3.0 g tumor. Anion-exchange chromatography using elution with a gradient of amm onium chloride was carried out as described previously [23].
108
The two small peaks observed at 0.49 and 1.03 ppm originate from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE), respectively [13-15]. The region of the spectrum to low field of Pi originates from phosphomonoesters. In other systems, this region has been assigned to sugar phosphates [9-11]. In many instances, these resonances are derived from various glycolytic intermediates [12,24,25]. In RIF-1 extracts, however, there is little or no evidence of these derivatives. Comparison with authentic samples shows little, if any, of the common hexose and triose phosphates. The most intense resonances in this spectral region emanate from phosphorylethanolanaine at 4.50 ppm and phosphorylcholine at 3.99 ppm. Because of a possible overlap with ribose 5-phosphate, phosphorylethanolamine was further assigned by selectively irradiating the OCH 2 proton resonance (3.99 ppm) (see below) and observing the collapse of the JP-H coupling (6.10 Hz) in the 31p-NMR spectrum. The presence of both phosphorylethanolamine and phosphorylcholine were confirmed by automated phosphate analysis (Fig. 2
TABLE II A U T O M A T E D P H O S P H A TE ANALYSIS OF A RIF-1 T U M O R P E R C H L O R I C A C I D EXTRACT Perchloric acid extract was prepared from a 6.0 g tumor that was frozen after surgical excision from the anesthetized mouse. Analysis was performed on a 0.2 ml sample corresponding to 0.6 g of tumor tissue. Peak
A mount ( n m o l / g )
ATP A D P + UTP Pi Phosphorylethanolamine phosphorylcholine N A D + hexose phosphate AMP Triose phosphate Fructose 1,6-bisphosphate + 2,3-diphosphoglycerate CTP UMP Phosphocreatine
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Fig. 3. 31p-NMR spectrum (162 MHz) of a perchloric acid extract of cultured R I F - I cells grown in Waymouth medium 752/1. The sample, contained in 2.0 nil, was prepared from 0.3 ml of packed cells. The pH of the sample was 6.8 and the spectrum was obtained in 3500 scans. The spectral conditions are as described in the legend to Fig. 1 for the extract spectrum. Abbreviations, same as fig 1.
109 and Table II). Under the conditions used, GPC and G P E could not be determined, since they were not retained on the anion-exchange column. The relatively low concentrations of ATP in this sample may reflect, in part, the advanced stage of growth of this tumor. In 31p spectra of RIF-1 extracts the phosphorylethanolamine peak is substantially more intense than the phosphorylcholine peak, whereas glycerophosphorylcholine appears to be more abundant than glycerophosphorylethanolamine. In extracts of other tumors (Dunn osteosarcoma [4], MOPC 104D myeloma and human colon xenograft (CX-1); unpublished results), the opposite trend appears to hold, indicating that differences may be specific for the type of tumor examined or they may reflect variations in the metabolic state of the tumors. Some RIF-1 extracts exhibited small, vestigial A M P and glucose 6-phosphate resonances, but these peaks were absent or below the noise level in spectra of most tumor extracts. The 31p spectrum of a perchloric acid extract of RIF-1 tissue culture cells is shown in Fig. 3. The in vitro t u m o r extract lacks the p h o s p h o r y lethanolamine peak that was prominent in the spectrum of the in vivo extract. The most intense phosphomonoester resonance originates from phosphorylcholine. The spectra of the perchloric acid extract of the isolated cells contained an additional unidentified resonance (tentatively assigned to AMP) to low field of the phosphorylcholine peak. Except for these differences, the 31p spectrum of the in vitro RIF-1 tumor resembles that of the in vivo tumor. The only major unassigned 31p resonance is a complex peak centered at - 1 2 . 1 3 ppm which is generally attributed to diphosphodiesters. On the basis of chemical shifts, we have ruled out the possibility that this peak arises from cytidine diphosphocholine or cytidine diphosphoethanolamine.
1H-NMR spectrum A typical ~H-NMR spectrum of the perchloric acid extract of a RIF-1 tumor in 2H20 is shown in Fig. 4. Well-resolved resonances of various amino acids, nucleotides, creatine, phosphocreatine, phosphorylethanolamine, phosphorylcholine, GPE, G P C and lactate are observed. Coupled res-
onances were identified by shift-correlated two-dimensional N M R spectroscopy (Fig. 5) [20,21]. The pH titration behavior of various resonances was instrumental in their assignment to specific species. The phosphocreatine and creatine methyl singlets near 3.0 ppm can be resolved from each other below pH = 7.8 (creatine is at higher field). The methylene 1H resonances of these species yield singlets near 4.0 ppm that can be resolved over the entire pH range. Hence, both components of the creatine pool can be monitored by 1H-NMR. The methyl peaks of phosphorylcholine, glycerophosphorylcholine and possibly choline are resolved singlets at pH > 4.8 with a chemical shift of about 3.2 ppm. At lower pH, these peaks merge. In a similar manner, the N C H 2 hydrogens of these species give rise to resolvable resonances near 3.6 ppm, and the OCH 2 hydrogens yield resolvable peaks near 4.2 ppm at pH > 5. A complex multiplet from the glycerol hydrogens of GPC occurs near 3.9 ppm. The most intense 1~ resonances of the tumor extract originate from taurine (2-aminoethanesulfonic acid). The presence of this compound as well as of other amino acids in tumor extracts was verified by amino-acid analysis. The region of the spectrum near 3.2 ppm is very complex as a result of overlap of resonances emanating from the taurine SCH2, phosphorylethanolamine N C H 2 and the phosphorylcholine and glycerophosphorylcholine N C H 3 hydrogens. The lactate and threonine CH 3 peaks overlap near neutral pH but are resolved in acid or alkaline solution. The low-field portion of the 1H spectrum (Fig. 4b) is less complex than its high-field counterpart and shows resonances derived primarily from adenine derivatives and some uracil- and cytosine-containing compounds. Also present were resonances of low intensity arising from phenylalanine, tyrosine and histidine or their derivatives. It should be emphasized that this portion of the spectrum has been amplified 16-fold relative to the high-field portion, and that the species giving rise to low-field resonances are present at significantly lower concentrations than those producing the more intense high-field resonances. Although qualitatively similar to the extract of the in vivo tumor, the extract of RIF-1 tissue culture cells shows some quantitative differences.
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Fig. 4. The 1H-NMR spectrum (400 MHz) of the perchloric acid extract prepared from a 7.8 g RIF-1 tumor showing (a) the high-field and (b) the low-field regions. The amplitude of the peaks in (b) is 16-times that of the peaks in (a). The p H of the sample was 6.8. Spectral conditions employed: 16K data points, 90 o flip angle, 3.05 s recycle time, 300 scans, no solvent suppression.
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Fig. 6. Portion of a 13C-NMR spectrum (100 MHz) of the RIF-1 tumor extract described in Fig. 5. The pH of the extract was 6.8. Conditions: 90 o flip angle; 3.2 s recycle time; 10000 scans. TSPext, external reference, 4,4-dimethyl-2,2,3,3-tetradeutero-4-silapentanoate.
The major difference is the lower concentrations of creatine, phosphocreatine and taurine. Several 1H resonances remain unidentified. At pH 9.1, two relatively intense coupled triplets are resolved at 3.07 and 3.42 ppm which arise from an unstable, unsymmetrically 1,2-substituted ethane
T A B L E III SELECTED C H E M I C A L SHIFTS F O R 13C R E S O N A N C E S IN A RIF-1 E X T R A C T (pH 6.8) Compound
Chemical shift (ppm)
Taurine Lactate phosphorylethanolamine phosphorylcholine Phosphocreatine Creatine Glycine Alanine Glutamic acid
38.18, 50.23 (CH2-N , CH2-S ) 22.86, 71.19 (CH3, CH) 43.37, 62.93 (CH2-N, CH2-O ) 56.73 ((CH3)3) 39.59, 60.76 (CH 3, CH2) 39.79, 56.60 (CH3, CH2) 44.24 (CH2) 18.92, 53.27 (CH 3, CH) 29.65, 36.19, 57.32 (CH2-CH, CH 2-CO, CH-CO)
derivative (i.e., observed only in flesh extracts). We have confirmed that these peaks do not originate from 2-aminoethanethiol, isethionic acid or hypotaurine.
13C_NMR The 13C-NMR spectrum of an extract from a single very large (7.8 g) tumor was obtained in an overnight experiment (Fig. 6, Table III). As in the 1H-NMR spectrum, the most intense resonances (at 38.18 and 50.23 ppm) originate from taurine. Resonances of other amino acids - alanine, glycine and glutamic acid - are observed as are resonances originating from lactate, phosphorylethanolamine, phosphorylcholine, creatine and phosphocreatine. A few resonances remain to be identified, including a fairly intense peak at approx. 163 ppm (not shown). There was a great deal of variability in the samples obtained from different tumors. Variables such as tumor size, degree of necrosis, hypoxic cell fraction, etc., all have profound effects on the metabolic state of tumors. Since the major objec-
113 tive of this paper was to identify metabolite resonances, no attempt was made to grade the tumors used in this study by size or time post-implantation, or to systematically investigate these variations. Discussion
Several N M R studies have been directed toward the identification of phosphorus metabolites in tumor cells in culture [12-15], including studies by Evans and Kaplan [12] who have extended such work to extracts of some solid human tumor xenografts in athymic mice. The 31p spectra of these intact, dispersed tumor cells all exhibit Pi, N T P and N D P resonances. However, considerable differences were observed in the phosphomonoester and diester resonances, and in the composition of the nucleotide pool. Thus, extracts of H e L a cells [12] like those of RIF-1 tumors exhibited two sets of resonances corresponding to purine and pyrimidine nucleotides, whereas extracts of Ehrlich ascites cells [14] exhibited only the purine nucleotide resonances. In the case of RIF-1 tumors, A T P was the predominant high-energy phosphate in the cell (Tables I and II); however the presence of other nucleotides demonstrated that, in general, care must be taken in attributing N T P and N D P resonances exclusively to adenine derivatives. Some ambiguity has existed concerning the identity of the phosphomonoester resonances. Evans and Kaplan [12] did not identify these resonances in their study of solid human tumor xenografts in athymic mice. In spectra of in vivo tissues these resonances have generally been assigned to sugar phosphates [9-11]. In instances where resonances of sugar phosphates have been observed, changes in these peaks have been attributed to anaerobic glycolysis [25]. However, N a v o n et al. [15] reported that virus-transformed lymphoid cell lines exhibited 31p spectra containing mostly phosphorylethanolamine and phosphorylcholine in the sugar phosphate region. We have found that for in vivo RIF-1 tumors, the phosphomonoester resonances, like those originating f r o m cultured virus-transformed lymphoid cells [15], arise almost exclusively from phosphorylethanolamine and phosphorylcholine, and glycolytic intermediates are usually below the level of N M R detection. These observations, to-
gether with similar findings on two other tumors (Dunn osteosarcoma and M O P C 104E myeloma) that we have examined, suggest that processes other than glycolysis are responsible for changes in the intensity of the phosphomonoester resonances of at least some in vivo tumors, although glycolysis does occur in these tumors as is demonstrated by the high level of lactic acid (Fig. 4a) [26]. We considered the possibility that such glycolytic intermediates may have been rapidly depleted by the trauma associated with isolation of the tumor. However, the small differences between spectra of tumors extracted after excision from the in vivo host, from the frozen host and from the freeze-clampled tumor argue against this possibility. Agreement between the chemical shifts of these resonance in spectra of the in vivo tumor and of tumor extracts further supports this conclusion. In this regard, RIF-1 tumors appear to be comparatively stable in their maintenance of high levels of phosphorus metabolites. Ribose 5-phosphate has been reported as a major metabohte in 31p-NMR spectra from guinea-pig brain extracts [27]. Our studies of this compound rule it out as a possible 31p-NMR-detectable metabolite in the RIF-1 tumor. It should be noted, however, that the chemical shifts and p H titration behavior of ribose 5-phosphate and phosphorylethanolamine are strikingly similar. We have found that for authentic samples, their chemical shifts coincide at p H greater than 7.7 and less than 4.6. They can easily be distinguished, however, by specific heteronuclear 1H decoupling experiments, which have been performed in our study but have not been reported in the study of guinea-pig brain extracts. The in vitro tumor cells did not exhibit either a ribose 5-phosphate or a phosphorylethanolamine 31p resonance, but did show a phosphorylcholine peak. The presence of choline but not ethanolamine in the growth medium could explain the presence of phosphorylcholine but not phosphorylethanolamine in the in vitro cell preparation. There is some controversy about the origin of the phosphorylcholine, phosphorylethanolamine, G P C and G P E resonances in spectra of various intact tissues [15,28-30]. Decomposition of phospholipids in regions of tumor necrosis could generate some or all of these metabolites. Alternatively, these molecules may accumulate in regions of rapid
114 tumor growth to meet the demands for biosynthesis of phospholipids. Both of these processes are probably occurring in the in vivo tumor, but it is important to determine their relative contributions to elevated levels of these metabolites. The fact that these resonances increase with increasing tumor size (and hence with decreasing growth fraction of the tumor) suggests that the catabolic mechanism is playing the predominant role in generating these metabolites. If this proves to be the case, then levels of these derivatives may serve as indices of the' extent of tumor necrosis. There has been also considerable controversy whether tumor cells produce sufficient phosphocreatine for detection by 3~p-NMR or whether phosphocreatine resonances originate from normal cells infiltrating or adjacent to the tumor. Spectra of a variety of cells in culture [14,15] and of a Walker sarcoma implanted in a rat [5] did n o t exhibit a phosphocreatine peak. However, spectra both of other subcutaneously implanted murine tumors [1-4] and of other tumor cells in culture [12,32] demonstrated the presence of phosphocreatine. The fact that phosphocreatine was detected by some investigators and not by others, in some cases for the same in vitro cell lines (either HeLa or Ehrlich ascites), suggests that environmental factors may be responsible for these differing results. In vivo studies of brains [33] and hearts [34] clearly demonstrate that even mild hypoxia is sufficient to convert phosphocreatine to creatine. At the high cell densities employed in the in vitro studies (approx. 1.108 cells/ml), some degree of hypoxia may be expected even if gases are bubbled through the cell suspension. Other effects such as accumulation of various toxic metabolites may also affect the phosphocreatine/creatine ratio. The recent study of Agris and Campbell [35] reports the presence of phosphocreatine in the aH - N M R spectrum of intact Friend leukemia cells and of their perchloric acid extracts. Since the chemical shift differences of phosphocreatine and creatine are small and sensitive to pH, it is not clear how these authors ruled out creatine as the resonance observed. This possibility is suggested by the reported absence of phosphocreatine in 3~p-NMR spectra of these cells [15]. An alternative explanation that phosphocreatine originates only from normal tissues either
proximal to or embedded in the tumor is untenable (at least in the case of the RIF-1 tumor), in view of the observation that perchloric acid extracts from pure cultures of RIF-1 cells (Fig. 3) exhibit a phosphocreatine resonance. Furthermore, r.f. field plots (with phantom samples) of the probe employed in this study clearly exclude any spectral contributions from tissues outside the tumor, a fact that was further substantiated by the absence of signals when a tumor-free mouse was placed in the N M R probe. Some blood vessels and fibrous tissues are present in the tumor and may contribute to the observed spectral resonances, but histological examination indicates that these cannot account for the majority of the spectral intensity. Lymphocyte infiltration is also known to be minimal in this nonimmunogenic tumor [17]. Whether the observed phosphocreatine results from endogenous or exogenous creatine remains to be determined.It may be that, as has been reported for Ehrlich ascites cells [32], RIF-1 tumors lack the ability to synthesize creatine, but retain the ability to phosphorylate it. In some RIF-1 extracts, an extra 31p resonance was observed approx. 0.1 ppm to low field of the phosphocreatine resonance. This signal results from coupling of the phosphorus nucleus to protons on the phosphoramide nitrogen under conditions where not all of the H 2 0 was replaced by 2H20 [36]. Because of its approx. 16-fold greater sensitivity compared to 3~P-NMR, ~H-NMR may prove to be particularly useful for clinical applications. This nucleus offers the additional advantage of directly monitoring several metabolites that cannot be readily detected by other N M R techniques. Noteworthy among these is lactate, whose elevated levels in tumors is well documented [37], and may prove useful in the diagnosis of cancer. Near neutral pH, the lactate methyl resonance overlaps with the threonine methyl peak. Overlap can be eliminated in acid solution, but since lactate is usually much more abundant than threonine, little error is introduced by ignoring this overlap. Taurine has been detected in a variety of normal and neoplastic tissues [38]. This aminosulfonic acid, which is produced from the catabolism of cysteine, exists in tissues as a free amino acid and is not known to be utilized for protein synthesis or i
115
as a source of energy. To date, the best known function of this metabolite is its conjugation with steroids in the biosynthesis of bile acids. While taurine has generally been considered nonessential, there is evidence that it is required for normal development of the retina [39]. Its role and the reason for the substantial decrease of taurine levels in the in vitro tumor remain to be determined. However, the observation of lower levels of this and other metabolites (such as creatine and phosphocreatine) in cultured cells suggests that the host plays a considerable role in determining the levels of some metabolites found in tumors. The 13C-NMR method has proven useful for analysis of metabolic pathways by monitoring the distribution of carbon atoms that have been enriched with this isotope [40]. Natural abundance 13C-NMR, which has been employed in studies of intact excised muscle [41] and in the present study of RIF-1 tumor extracts, is of limited utility because of the low sensitivity of this nucleus and the consequent requirement for long accumulation times that are impractical for in vivo applications. However, these studies demonstrate that the method is quite useful for monitoring a number of specific metabolites in the extract under conditions of minimal spectral overlap. For example, discrete ~3C resonances of lactate can be monitored without the overlap problems encountered with this metabolite in the proton N M R spectrum (see above). Resonances of taurine and glutamic acid are also better resolved in the ~3C- than in the H - N M R spectrum. These studies demonstrate the need for the unambiguous assignment of resonances from in vivo N M R spectra of tissues. The wealth of information available from multi-nuclear N M R (especially 1H) is also clearly evident. It will probably be possible to obtain at least some of this information in clinical N M R examinations of tumors and other tissues.
Acknowledgements This work was supported by USPHS Grant CA13148 (A.F. LoBuglio and J.D.G.), CA-36346 (J.D.G.), CA 33304 (J.D.G.), and Faculty Research Grant FRA-162 (J.D.G.) from the American Cancer Society. We thank Dr. Robert F. Kall-
man of Stanford University for supplying us with the RIF-1 tumor cells. N.R.K. is a Leukemia Society of America Scholar, 1982-1987.
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