Dual-Frequency Proton Spin Relaxation Measurements on Tissues From Normal and TumorBearing Mice' Barry A. Coles 2 , 3 ABSTRACT-Measurements of pulsed nuclear magnetic resonance relaxation times T, and T2 were made at 2.7 MHz and 15 MHz on water protons in liver, kidney, spleen, muscle, and brain tissue from normal A.SW mice, and in the same tissues and tumors from A.SW mice developing MSWBS tumors (an ascites sarcoma) following dorsal sc implantation of tumor fragments. The measurement precision obtained from improved spectrometer design made it possible to show that T, and T2 in all tissues except brain were increased by the presence of the tumor in the animal. The responses exhibited by T, and T2 in liver and kidney were proportional to the size of the tumor. The smaller responses shown by T, in spleen (15 MHz) and T, and T2 in muscle (2.7 MHz) also showed a significant correlation with tumor size. The relaxation times for tumor (T, at2.7 MHz, T2 at2.7 and 15 MHz) showed a significant negative correlation with tumor size: The times decreased as tumor size increased. The results were analyzed by use of the two-phase fast exchange model and were consistent with the effects expected if tissue water content increased and tumor water content decreased as tumor size increased. The analysis indicated that the effects arose primarily through changes in b, the fraction of water bound to fast exchange sites on the protein, with important modifications from changes in the correlation times T c and T m ; T c controlled the frequency that must be chosen for specific diagnostic applications.-J Nat! Cancer Inst 57: 389-393, 1976.
Some nonmalignant tissues in tumor-bearing animals exhibit increased values of the proton spin relaxation time T 1 (1-3), and an increased water content is also associated with the increased values oi T', (4). Whetherand how-the increased water content actually causes the T I increase does not appear to have been established. No tumor-induced effects on T 2 seem to have been confirmed. T I has mainly been measured in the 15to 60-MHz region, but theoretical prediction based on dispersion measurements (5) suggests that greater responses might be obtained at lower frequencies in the 2MHz region, which could assist the development of diagnostic applications of NMR. The possibility of extending spin-mapping techniques (6) to give in vivo whole body scanning for tumors by NMR also emphasizes the importance of low-frequency measurements, inasmuch as economic considerations preclude the use of high magnetic fields, and hence high NMR frequencies, over such large volumes. The recent development of two pulsed NMR spectrometers of an improved design (7), one operating at 2.7 MHz, has made it possible to investigate the frequency dependence of the tumor-induced T I response and to examine the behavior of T 2 with far greater accuracy than has previously been possible. MATERIALS AND METHODS
VoL. 57, NO.2, AUGUST 1976
ABBREVIATION USED:
NMR=nuclear magnetic resonance.
, Received November 14, 1975; accepted January 16, 1976. Physical Chemistry Laboratory, Oxford University, South Parks Road, Oxford OXI 3QZ, England. 3 1 am indebted to Professor H. Harris for the gift of mice and tumor materials, to Mr. J. H. D. Kent for preparing the tumorbearing animals, to Newport Instruments Ltd. for the loan of the 2.7MHz pulsed NMR spectrometer, and to Professor M. M. Pintar for valuable discussions. 2
We used A.SW female mice (Sir William Dunn School of Pathology, Oxford, England) 53-83 days old and weighing 20-25 g at the time of measurement. MSWBS tumors (an ascites sarcoma originally derived from a 3-
Downloaded from https://academic.oup.com/jnci/article-abstract/57/2/389/910143 by University of Durham user on 07 March 2018
methylcholanthrene-induced tumor) (8) ranging from 0.25 to 1.6 g were induced by dorsal sc implants of tumor fragments; 11-18 days were allowed for development. All mice were killed by cervical dislocation. Tissue samples were dissected, blotted, and placed in 2-ml sample tubes which were then closed and kept in ice. Measurements were completed within 6 hours of death; the samples were equilibrated to room temperature (l9± 1°C) immediately before being placed in the spectrometers. Corrections were not applied for temperature coefficients, since these were small compared with the differences between animals. Two pulsed NMR spectrometers with crossed-coil probes were used, one operating at 2.7 MHz and the other at 15 MHz. Both were equipped with circuits that provided automatic control of magnetic field, sample coil tuning, and pulse amplitude. The instrument measurements were converted directly to relaxation data by digital and analogue computing circuits connected to the outputs of the spectrometers. T I was determined with the use of 90°-90° pulse sequences over the portion of the recovery characteristic beginning 50 msec after the first 90° pulse and continuing for approximately T I to the nearest multiple of 50 msec. In this region recoveries approximated single exponential behavior and were treated as such. The free induction decays were sampled for 200 usee (250 usee at 15 MHz) beginning 150 usee after the end of the 90° pulse to avoid inclusion of signal from protons other than on water molecules. T 2 was determined by means of the Carr-Purcell Meiboom-Gill 90-nI80° sequence, with the 180° pulse spacing at 1 msec (2 msec at 15 MHz); samples of 200 usee (250 usee at 15 MHz) were taken at the appropriate echo peaks. The portion of the decay characteristic used at 2.7 MHz ran from 10 msec after the initial 90° pulse and continued for approximately T 2 to the nearest 10 msec; at 15 MHz the portion used ran from 4 msec after the 90° pulse and lasted 40 or 80 msec, whichever was nearer to T 2 • The decays were treated as single exponentials over this region. The slightly different arrangements at the two frequencies arise from the programmer logic designs in the two instruments and should not affect the conclusions drawn here. The 2.7-MHz sample coil accepted up to 2 ml of sample;' thus whole organs, tumors, or the muscle from both hind legs could be measured. Most samples were
389
J
NATL CANCER INST
390
COLES
less than 2 ml and caused some loss of measurement precision. A standard error of 2% was obtained for the smallest samples (spleen, weighing 70-100 mg), compared with 0.25-0.35% for the largest samples (muscle). The 15-MHz coil restricted samples to 0.2 ml, so that only subsamples of liver, brain, muscle, or tumor were measured. Differences were observed between subsarnpies from the same tissue, but these were negligible compared with the differences between animals. Standard errors at 15 MHz were 0.1-0.3%. RESULTS
The mean relaxation times for normal and tumorbearing animals are presented in table I, and the distribution of T 1 values is shown in the form of a dispersion map in text-figure 1. The value of T 1 at 2.7 MHz was plotted against the value at 15 MHz for each sample, and for each tissue two envelopes are shown: One encloses the points from normal animals and one encloses those from tumor-bearing animals. The envelopes were drawn to include the standard error of the points. The size and shape of the envelopes were determined primarily by the variation between animals, since (except for spleen at 2.7 MHz) the standard error of individual points was comparable with or less than the size of the symbols used for plotting.
Although the envelopes for muscle and spleen from tumor-bearing animals mainly appeared to be displaced from the healthy position, those for liver and kidney were elongated compared with their normal counterparts and suggested that a progressive variation of relaxation time occurred. Consequently, regressions were investigated; good correlations [P (result due to chance)
Some nonmalignant tissues in tumor-bearing animals exhibit increased values of the proton spin relaxation time T 1 (1-3), and an increased water content is also associated with the increased values oi T', (4). Whetherand how-the increased water content actually causes the T I increase does not appear to have been established. No tumor-induced effects on T 2 seem to have been confirmed. T I has mainly been measured in the 15to 60-MHz region, but theoretical prediction based on dispersion measurements (5) suggests that greater responses might be obtained at lower frequencies in the 2MHz region, which could assist the development of diagnostic applications of NMR. The possibility of extending spin-mapping techniques (6) to give in vivo whole body scanning for tumors by NMR also emphasizes the importance of low-frequency measurements, inasmuch as economic considerations preclude the use of high magnetic fields, and hence high NMR frequencies, over such large volumes. The recent development of two pulsed NMR spectrometers of an improved design (7), one operating at 2.7 MHz, has made it possible to investigate the frequency dependence of the tumor-induced T I response and to examine the behavior of T 2 with far greater accuracy than has previously been possible. MATERIALS AND METHODS
VoL. 57, NO.2, AUGUST 1976
ABBREVIATION USED:
NMR=nuclear magnetic resonance.
, Received November 14, 1975; accepted January 16, 1976. Physical Chemistry Laboratory, Oxford University, South Parks Road, Oxford OXI 3QZ, England. 3 1 am indebted to Professor H. Harris for the gift of mice and tumor materials, to Mr. J. H. D. Kent for preparing the tumorbearing animals, to Newport Instruments Ltd. for the loan of the 2.7MHz pulsed NMR spectrometer, and to Professor M. M. Pintar for valuable discussions. 2
We used A.SW female mice (Sir William Dunn School of Pathology, Oxford, England) 53-83 days old and weighing 20-25 g at the time of measurement. MSWBS tumors (an ascites sarcoma originally derived from a 3-
Downloaded from https://academic.oup.com/jnci/article-abstract/57/2/389/910143 by University of Durham user on 07 March 2018
methylcholanthrene-induced tumor) (8) ranging from 0.25 to 1.6 g were induced by dorsal sc implants of tumor fragments; 11-18 days were allowed for development. All mice were killed by cervical dislocation. Tissue samples were dissected, blotted, and placed in 2-ml sample tubes which were then closed and kept in ice. Measurements were completed within 6 hours of death; the samples were equilibrated to room temperature (l9± 1°C) immediately before being placed in the spectrometers. Corrections were not applied for temperature coefficients, since these were small compared with the differences between animals. Two pulsed NMR spectrometers with crossed-coil probes were used, one operating at 2.7 MHz and the other at 15 MHz. Both were equipped with circuits that provided automatic control of magnetic field, sample coil tuning, and pulse amplitude. The instrument measurements were converted directly to relaxation data by digital and analogue computing circuits connected to the outputs of the spectrometers. T I was determined with the use of 90°-90° pulse sequences over the portion of the recovery characteristic beginning 50 msec after the first 90° pulse and continuing for approximately T I to the nearest multiple of 50 msec. In this region recoveries approximated single exponential behavior and were treated as such. The free induction decays were sampled for 200 usee (250 usee at 15 MHz) beginning 150 usee after the end of the 90° pulse to avoid inclusion of signal from protons other than on water molecules. T 2 was determined by means of the Carr-Purcell Meiboom-Gill 90-nI80° sequence, with the 180° pulse spacing at 1 msec (2 msec at 15 MHz); samples of 200 usee (250 usee at 15 MHz) were taken at the appropriate echo peaks. The portion of the decay characteristic used at 2.7 MHz ran from 10 msec after the initial 90° pulse and continued for approximately T 2 to the nearest 10 msec; at 15 MHz the portion used ran from 4 msec after the 90° pulse and lasted 40 or 80 msec, whichever was nearer to T 2 • The decays were treated as single exponentials over this region. The slightly different arrangements at the two frequencies arise from the programmer logic designs in the two instruments and should not affect the conclusions drawn here. The 2.7-MHz sample coil accepted up to 2 ml of sample;' thus whole organs, tumors, or the muscle from both hind legs could be measured. Most samples were
389
J
NATL CANCER INST
390
COLES
less than 2 ml and caused some loss of measurement precision. A standard error of 2% was obtained for the smallest samples (spleen, weighing 70-100 mg), compared with 0.25-0.35% for the largest samples (muscle). The 15-MHz coil restricted samples to 0.2 ml, so that only subsamples of liver, brain, muscle, or tumor were measured. Differences were observed between subsarnpies from the same tissue, but these were negligible compared with the differences between animals. Standard errors at 15 MHz were 0.1-0.3%. RESULTS
The mean relaxation times for normal and tumorbearing animals are presented in table I, and the distribution of T 1 values is shown in the form of a dispersion map in text-figure 1. The value of T 1 at 2.7 MHz was plotted against the value at 15 MHz for each sample, and for each tissue two envelopes are shown: One encloses the points from normal animals and one encloses those from tumor-bearing animals. The envelopes were drawn to include the standard error of the points. The size and shape of the envelopes were determined primarily by the variation between animals, since (except for spleen at 2.7 MHz) the standard error of individual points was comparable with or less than the size of the symbols used for plotting.
Although the envelopes for muscle and spleen from tumor-bearing animals mainly appeared to be displaced from the healthy position, those for liver and kidney were elongated compared with their normal counterparts and suggested that a progressive variation of relaxation time occurred. Consequently, regressions were investigated; good correlations [P (result due to chance)
