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Acute pancreatitis in rats: A 31P nuclear magnetic resonance study
JOURNAL
OF SURGICAL
43, 172- 178 ( 1987)
RESEARCH
Acute Pancreatitis
in Rats: A 31P Nuclear Magnetic
Resonance
Study’
OFERKAPLAN, M.D.,* TAMMAR KUSHNIR, PH.D.,? URI SANDBANK,
M.D.,+ AND GIL NAVON, PH.D.t
‘Department of Surgery, Tel-Aviv Medical Center, Rokach Hospital, Tel-Aviv, Israel; tSchool of Chemistry, Tel-Aviv University, Tel-Aviv, Israel; and SCasper Institute of Pathology, Beilimon Medical Center, Petah-Tikva and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Submitted for publication January 23, 1986 High resolution ‘iP nuclear magnetic resonance (NMR) was used to evaluate the severity of acute pancreatitis in rats. Experimental pancreatitis was induced by intraparenchymal injection of 10% sodium taurocholate. Pancreases were removed at various time periods and the NMR spectrum of the whole organ was recorded. Metabolic changes taking place during the progression of the disease were measured and correlated with the pathologic changes. Gradual depletion of the high energy compounds, adenosine triphosphate and phosphocreatine, was observed. The NMR spectral changes paralleled the extension of the pathologic lesions and were found to constitute a reliable indicator of the severity of acute pancrcatitis. It is suggested that high resolution NMR may be used to evaluate the pathogenesis and therapy of various forms of experimental pancreatitis. 0 1987 Academic Rw., Inc.
INTRODUCTION
Acute pancreatitis is characterized by a broad pathologic spectrum ranging from minimal edema through hemorrhagic necrosis to irreversible damage to the gland [S, lo]. The relationship between clinical presentation, laboratory and diagnostic parameters, and pathologic processes is iIl defined. Aside from direct inspection of the pancreas during laparatomy or autopsy, there are no clear-cut objective and quantitative parameters to assessthe severity and prognosis of the disease (13). Such an assessment is essential for choosing the optimal therapeutic management and for studying the efficacy of the treatment. The levels of high energy phosphorous compounds have been reported to be reduced in pancreatic biopsies from experimental pancreatitis in dogs [3]. It was sug’ This work was supported in part by The Fund for Basic Research administered by The Israel Academy of Sciences and Humanities. It was presented in part at the Second Congress of European Society of Magnetic Resonance in Medicine and Biology, October 3-5, 1985. Montreux, Switzerland. 0022-4804/87 $1.50 Cwyrigbt Q 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
gested that this energy depletion reflects the progression of the disease from edematous to hemorrhagic status. High resolution “P nuclear magnetic resonance (NMR) spectroscopy offers a means to detect and measure the levels of phosphorous compounds in tissues and whole organs [4, 51. The technique of topical NMR (TMR) enables noninvasive continuous measurement of these levels. Thus, physiologic and pathologic processes can be continuously monitored in vivo [5]. We describe herein 31P NMR studies of intact pancreas taken from rats with experimentally induced pancreatitis. The 3’P NMR spectra of diseased pancreases were compared with the spectrum of the normal organ. The changes in the levels of high energy compounds, the appearance of pancreatitis-associated compounds, and the intracellular pH were detected and evaluated. NMR findings were correlated with pathological changes as defined by macroscopic and histologic examinations. To our knowledge these are the first studies employing 3’P NMR spectroscopy in clinical and experimental pancreatitis.
172
KAPLAN
MATERIALS
173
ET AL.: 3’P NMR OF PANCREATITIS
AND METHODS
Induction of pancreatitis. Female Sprague-Dawley rats weighing 200-300 g were given food and water ad libitum. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30-40 mg/kg). A small midline laparatomy was performed, and the stomach, duodenum, spleen, and pancreas were brought out through the incision. One milliliter of 10% sodium taurocholate (Sigma, lot 94F-5032) was manually infiltrated into the tail and body of the pancreas with a 25G needle. This method of pancreatitis induction is simple, predictable, and highly reproducible [ 1, 2, 61 and is therefore very suitable for NMR studies. Animals were reoperated at various time periods (between 20 and 360 min, see Table 1) at which time the pancreas was quickly removed and placed in chilled (0-4°C) phys-
iologic solution with oxygen bubbling in the NMR tube (10 mm diameter). The NMR spectrum of the whole pancreas was immediately recorded. Control rats were injected with saline solution into the pancreas. A total of 25 pancreases were examined (see Table 1). Preparation of extracts. In order to assign the 31P NMR signals, perchloric acid extracts of normal and sodium taurocholate-treated pancreases were made, as previously described [ 71. 3’P NMR studies. The NMR spectrometer was Bruker AM-360 WB operating at 145.78 MHz for “P measurements, equipped with a variable frequency IO-mm probe. The temperature was 2 f 1“C. The conditions of the NMR measurements of whole pancreases are described in the legend to Fig. 1. The 31P spectra of the extracts were recorded at various pH values. The titration
TABLE 1 THECORRELATIONSBETWEENTHETIMEELAPSEDFROMINDUCTIONOFPANCREATITIS,THENMR SPECTRALCHANGES,ANDTHEPATHOL~GICFINDINGS Interval from sodium taurocholate injection (mitt)
PH”
Normal (n = 4) Controld (n = 5) 20 25 30 30 30 45 45 60 75 100 120 135 140 140 180 360
7.20 + 0.06 7.20 + 0.06 7.09 7.08 6.92 7.24 7.08 7.24 7.08 7.09 7.19 7.02 7.11 7.22 7.30 7.11 7.17 7.13
PATP/Pi 0.7 2 0.2 0.7 + 0.2 0.14 0.07 0.09 0.2 0.21 0.49 0.12 0.13 0.13 0.05 0.14 0.05 0.09 0.09 0.15 0.08
Macroscopic damagec
Histologic damage ’
z ++ ++++ +++ +
z ++ ue up ue + Ue +++
!s ++ +++ +++ ++++ ++ ++++ ++++ ++++ ++ +++
LIPancreatic intracellular pH as determined by the chemical shift of inorganic phosphate. b The ratio between the intensity of the signals of ,!?ATP and inorganic phosphate. ‘The severity of the damage was graded from + to ++++. Izi = no damage. d 30, 50, 90, 120, and 160 min after saline injection. e u-Undetermined.
Ue
+++ Ue ++ ll= u’ ++++ Ue Lie
174
JOURNAL OF SURGICAL RESEARCH: VOL. 43, NO. 2, AUGUST
1987
the extension of necrotic and hemorrhagic areas and to the presence of saponification of fatty tissue within and around the pancreas. The histologic sections were reviewed by one of us (U.S.) without knowing the experimental conditions and the NMR spectrum of each pancreas. RESULTS
The NMR spectra of the pancreases were recorded at room temperature at the beginning of this study. The high energy phosphorous compounds, phosphocreatine (PCr) and adenosine triphosphate (ATP), are very labile under ischemic conditions, and our initial spectra contained virtually no PCr and minute amounts of ATP. Subsequent NMR studies were performed at low temperatures (2 f 1“C) with oxygen bubbling, resulting in 10.0 5.0 0.0 -10.0 -20.0 minimal PCr and ATP decomposition in the PPM NMR tube. FIG. 1. A series of j’P NMR spectra of whole rat panThe NMR spectrum of the intact normal creases before (a) and after (b-d) induction of pancreapancreas is shown in Fig. la. The signal at titis. = Interval after injection of sodium taurocholate, in minutes. Each spectrum is a collection of 400 scans 0.49 ppm was assigned as glycerophospho(duration 10 min) by applying 45” radio frequency choline (GPC) on the basis of its chemical pulses (13 psec) and relaxation delay of 1 sec. The spec- shift relative to external references and by its tra were collected with a 4K data points at a 7300-Hz (50 pH independence. GPC identification was ppm) spectral width. No proton decoupling was applied. verified by the addition of the genuine comFor detailed information regarding the assignment of pound to extracts where the NMR signal signals see text. coincided with the naturally occurring signal. All chemical shifts of phosphorous comcurves of their chemical shifts (6) were compounds in the NMR spectra of the pancreas puter fitted and the resultant values of acidic were calibrated using GPC as an internal 6, basic 6, and pK were compared with those standard at 0.49 ppm [9]. The signal of inorof known genuine compounds. Each spec- ganic phosphate (Pi) appeared in the region trum of the extract was a result of 600 scans 2.5-2.7 ppm; its exact chemical shift was of pancreatic inwith 45” pulses and relaxation delays of 1 used for the determination tracellular pH. Two peaks appeared in the sec. Proton decoupling was applied. Pathologic examination. After removal phosphomonoester region: a prominent one each pancreas was macroscopically examat about 4.2 ppm and another at about 3.7 ined for the presence of edema, hemorppm. These two peaks were identified by their chemical shifts and pK’s, as determined rhages, and necrotic areas. Following the NMR measurements the in extracts, as phosphoethanolamine (PEA) whole pancreas was fixed in buffered formaand phosphorylcholine (PCho), respectively. lin for 24 hr and embedded in paraffin. The signal of PCr (-2.5 ppm) was smaller Seven-micron-thick longitudinal sections of than the three signals of ATP (-5.1, - 10.0, the whole pancreas were stained with hema- 19.1 ppm) (see Discussion). The two peaks toxylin and eosine. The intensity of the le- at - 10.8 and - 12.3 ppm correspond to disions was evaluated and graded according to phosphodiester compounds, such as uridine I
t
KAPLAN
ET AL.: 3’P NMR OF PANCREATITIS
diphosphoglucose (UDPG). Control animals exhibited no changes in the spectra of pancreases removed 30, 50, 90, 120, and 160 min afier saline injection. Their spectra were practically identical to the spectrum of the normal pancreas shown in Fig. la. Sodium taurocholate-treated pancreases exhibited NMR spectral changes that became more pronounced as the disease progressed (Figs. lb, c, and d). The principal changes were gradual diminution of PCr and ATP and a concomitant increase of Pi. Thirty minutes after induction of pancreatitis there was only a slight decrease of PATP and a slight increase of Pi (Fig. lb). These changes were more pronounced 60 min after injection (Fig. lc), and virtually all high energy compounds disappeared after 140 min (Fig. Id). The NMR spectral changes correlated better with the macroscopic and microscopic features of the examined pancreas than with the time elapsed from sodium taurocholate injection. This was evident in several pancreases where minor spectral changes 2 and 3 hr after induction of pancreatitis were associated with minimal pathologic damage. It is noteworthy that while the PCr peak was initially smaller than ATP peaks, it did not completely disappear before ATP depleted (see Discussion). With the development of pancreatitis, a new peak appeared in the phosphodiester region, at -0.16 +. 0.04 ppm. This peak was found only in the early phases of pancreatitis and disappeared later when signs of severe damage to the organ predominated. It was found both in the whole organ and in extracts of pancreases treated with sodium taurocholate, indicating that the signal corresponds to a small phosphorus-containing molecule. The intracellular pH of the normal and control pancreases, as determined by the Pi chemical shifts, was in the range 7.14-7.26. In deseased pancreases only a slight pH decrease was found, in the range 6.92-7.30. However, pH changes were not progressive and did not parallel the progression of the disease.
175
No changes in the GPC signal were found in the early stages of pancreatitis. In the more severe stages some pancreases showed a marked depletion of GPC (Fig. Id), while others showed no change. Macroscopic appearance. Pancreases from control animals receiving saline injection showed no gross lesions, edema, or hemorrhages. The pancreas illustrated in Fig. lb appeared edematous with minimal hemorrhages. Extensive macroscopic damage was observed in the pancreas which had the most dramatic NMR spectral changes (Fig. Id). Histologic Jindings. The control pancreases showed a normal appearance of the lobules and surrounding fatty tissue (Fig. 2). The pathologic changes associated with mild NMR spectral changes consisted of few necrotic pancreatic lobules and mild saponification of the intralobular fatty tissue (Fig. 3). The severely affected animals (NMR spectrum-Fig. Id) showed nearly complete necrosis of the pancreatic parenchyma and severe fat necrosis in the intra- and peripancreatic fatty tissue (Fig. 4). Table 1 presents the correlations between the time elapsed from pancreatitis induction, the NMR spectral changes, and the pathologic lesions. The ratio of PATP to Pi is an index of the high energy compounds levels. The best correlation of the actual pathologic damage is with NMR spectral changes of these high energy compounds. DISCUSSION
The study described here points to the applicability of NMR spectroscopy for the quantitative measurements of metabolic changes that occur during acute pancreatitis and for the assessment of the severity of the disease. Such objective parameters are essential for both clinical evaluation and experimental research. Our data show a remarkable change in the “P NMR spectrum of the rat pancreas after the induction of pancreatitis, which correlated with the clinical course of the disease. The most significant changes were the disappearances of the high energy
176
JOURNAL OF SURGICAL
RESEARCH: VOL. 43, NO. 2, AUGUST
1987
FIG. 2. Saline injected control pancreas. Preservation of pancreatic glands and the islands of Langerham. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. 1a.)
compounds ATP and PCr. These spectral changes were found to be better indicators of the actual damage than the time elapsed from the sodium taurocholate injection.
The end result of the pathogenetic processes of acute pancreatitis is enzymatic destruction of pancreatic cells [lo]. Since high energy phosphorous compounds are essen-
FIG. 3. Mild necrotic changes. Few pancreatic lobules are necrotic. Peripancreatic fatty tissue shows fat necrosis. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. 1b.)
KAPLAN
ET AL.: “P NMR OF PANCREATITIS
FIG. 4. Severe necrotic changes. The pancreas is totally necrotic. Fatty tissue shows severe necrosis and saponification. Inflammatory cells around necrotic pancreas and fat. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. Id.)
tial in preserving cell integrity, their depletion in the presence of pancreatic cell necrosis is expected. In the early phases of the disease only edema and minimal necrosis were found, and the NMR spectrum showed a minimal change. In the more severe stages the histologic picture showed extensive necrosis, and in the concomitant NMR spectra virtually all high energy compounds disappeared. These findings support the hypothesis of Becker et al. [3] that the progressive falls of ATP and PCr are signposts of the progression from edematous reversible to necrotizing irreversible pancreatitis. As shown in the present study, these signposts can be detected by 31P NMR. The pathologic damage in acute pancreatitis spreads from local necrosis in the early phases toward diffuse necrosis in the advanced states [8, lo]. This process was demonstrated in the present study by the fact that PCr did not completely disappear before ATP depleted, as would be expected on biochemical grounds if the whole organ was simultaneously damaged. There is a major ad-
vantage in measuring the whole intact organ in one examination, as is done by NMR, in comparison with techniques that require biopsy samples and measure only a limited portion of the organ. The 31P NMR spectrum of the normal pancreas is governed by signals in the phosphomonoester region, which were identified as PEA and PCho, and there is a high peak of GPC in the phosphodiester region. PEA showed no change during acute pancreatitis, while GPC was depleted in some severely damaged pancreases concomitant with a slight increase in PCho. No definitive conclusion can be drawn as these results were not reproducible. The new peak which appeared at about -0.16 ppm in the early stages of pancreatitis is not yet identified. The appearance of this peak both in whole organs and extracts indicates that it is a low-molecular-weight compound, presumably a degradation product of membranous phospholipids. Hypothesizing that it may be of diagnostic value as a marker, we are pursuing the assignment of
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the signal in the hope that it may contribute to understanding the pathogenesis of pancreatitis. Since our experiments were conducted under relatively ischemic conditions, the concentrations of PCr in our normal pancreas must have been lower than those in the live organ. This fact is of minor importance in the present study, since all the pancreases were examined under identical conditions, and conclusions were drawn from comparative rather than absolute values. This principle of comparative studies is also the basis for the validity of the histologic examinations. Moreover, as the pancreases were in chilled physiologic solution throughout NMR measurements, spontaneous autolysis was negligible. We are currently performing 3’P NMR studies of perfused rat pancreas in a study on the evolvement of various forms of pancreatitis [ 1, 11, 121 and on the efficacy of different therapeutic approaches. The results of the present study constitute, in our opinion, a firm basis for future clinical studies with the aid of topical NMR to evaluate the severity and prognosis of acute pancreatitis. REFERENCES 1. Aho, H. J., Nevalainen, T. J., and Aho, A. J. Experimental pancreatitis in the rat. Eur. Surg. Res. 15: 28, 1983.
1987
Bawnik, J. B., Orda, R., and Wiznitzer, T. Acute necrotizing pancreatitis: An experimental model. Dig. Dis. Sci. 19: 1143, 1974. 3. Becker, H., Vinten-Johansen, J., Buckberg, G. D., and Bugyi, H. I. Correlation of pancreatic blood flow and high energy phosphates during experimental pancreatitis. Eur. Surg. Res. 14: 203, 1982. 4. Cohen, J. S., Knop, R. H., Navon, G., and Foxall, D. Nuclear magnetic resonance in biology and medicine. Ll$ Chem. Rep. 1: 368, 1983. 5. Gadian, D. G. Nuclear Magnetic Resonance and Its Applications to Living Systems. New York: Oxford Univ. Press, 1982. Pp. l-70. 6. Hadas, N., Orda, R., Orda, S., Bawnik, J. B., and Wiznitzer, T. Experimental acute pancreatitis in rats caused by intraparenchymal injection of sodium taurocholate. Isv. J. Med. Sci. 19: 194, 1983. I. Lawry, 0. H., and Passoneau, J. V. A Flexible System of Enzymatic Analysis. New York: Academic Press, 1972. Pp. 120-124. 8. Longnecker, 0. S. Pathology and pathogenesis of diseases of the pancreas. Amer. J. Pathol. 107: 103, 1982. 9. Navon, G., Ogawa, S., Shulman, R. G., and Yamane, T. “P nuclear magnetic resonance studies of Ehrlich ascites Tumor cells. Proc. Natl. Acad. Sci. USA 14: 87, 1917. 10. Robbins, S. L., Cortan, R. S., and Kumar, V. (Eds.) Pathologic Basis of Disease. 3rd ed. Philadelphia: Saunders, 1984. Pp. 963-965. 11. Saharia, P., Margolis, S., Zuidema, G. D., and Cameron, J. L. Acute pancreatitis with hyperlipemia: Studies with an isolated perfused canine pancreas. Surgery 82: 60, 1977. 12. Sanfey, H., Bulkley, G. B., and Cameron, J. L. The pathogenesis of acute pancreatitis. Ann. Surg. 201: 633, 1985. 13. Silen, W., and Steer, W. L. Pancreas. In S. I. Schwartz (Ed.) Principles of Surgery, 4th ed. New York: McGraw-Hill, 1984. Pp. 1350-1353. 2.
OF SURGICAL
43, 172- 178 ( 1987)
RESEARCH
Acute Pancreatitis
in Rats: A 31P Nuclear Magnetic
Resonance
Study’
OFERKAPLAN, M.D.,* TAMMAR KUSHNIR, PH.D.,? URI SANDBANK,
M.D.,+ AND GIL NAVON, PH.D.t
‘Department of Surgery, Tel-Aviv Medical Center, Rokach Hospital, Tel-Aviv, Israel; tSchool of Chemistry, Tel-Aviv University, Tel-Aviv, Israel; and SCasper Institute of Pathology, Beilimon Medical Center, Petah-Tikva and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Submitted for publication January 23, 1986 High resolution ‘iP nuclear magnetic resonance (NMR) was used to evaluate the severity of acute pancreatitis in rats. Experimental pancreatitis was induced by intraparenchymal injection of 10% sodium taurocholate. Pancreases were removed at various time periods and the NMR spectrum of the whole organ was recorded. Metabolic changes taking place during the progression of the disease were measured and correlated with the pathologic changes. Gradual depletion of the high energy compounds, adenosine triphosphate and phosphocreatine, was observed. The NMR spectral changes paralleled the extension of the pathologic lesions and were found to constitute a reliable indicator of the severity of acute pancrcatitis. It is suggested that high resolution NMR may be used to evaluate the pathogenesis and therapy of various forms of experimental pancreatitis. 0 1987 Academic Rw., Inc.
INTRODUCTION
Acute pancreatitis is characterized by a broad pathologic spectrum ranging from minimal edema through hemorrhagic necrosis to irreversible damage to the gland [S, lo]. The relationship between clinical presentation, laboratory and diagnostic parameters, and pathologic processes is iIl defined. Aside from direct inspection of the pancreas during laparatomy or autopsy, there are no clear-cut objective and quantitative parameters to assessthe severity and prognosis of the disease (13). Such an assessment is essential for choosing the optimal therapeutic management and for studying the efficacy of the treatment. The levels of high energy phosphorous compounds have been reported to be reduced in pancreatic biopsies from experimental pancreatitis in dogs [3]. It was sug’ This work was supported in part by The Fund for Basic Research administered by The Israel Academy of Sciences and Humanities. It was presented in part at the Second Congress of European Society of Magnetic Resonance in Medicine and Biology, October 3-5, 1985. Montreux, Switzerland. 0022-4804/87 $1.50 Cwyrigbt Q 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
gested that this energy depletion reflects the progression of the disease from edematous to hemorrhagic status. High resolution “P nuclear magnetic resonance (NMR) spectroscopy offers a means to detect and measure the levels of phosphorous compounds in tissues and whole organs [4, 51. The technique of topical NMR (TMR) enables noninvasive continuous measurement of these levels. Thus, physiologic and pathologic processes can be continuously monitored in vivo [5]. We describe herein 31P NMR studies of intact pancreas taken from rats with experimentally induced pancreatitis. The 3’P NMR spectra of diseased pancreases were compared with the spectrum of the normal organ. The changes in the levels of high energy compounds, the appearance of pancreatitis-associated compounds, and the intracellular pH were detected and evaluated. NMR findings were correlated with pathological changes as defined by macroscopic and histologic examinations. To our knowledge these are the first studies employing 3’P NMR spectroscopy in clinical and experimental pancreatitis.
172
KAPLAN
MATERIALS
173
ET AL.: 3’P NMR OF PANCREATITIS
AND METHODS
Induction of pancreatitis. Female Sprague-Dawley rats weighing 200-300 g were given food and water ad libitum. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30-40 mg/kg). A small midline laparatomy was performed, and the stomach, duodenum, spleen, and pancreas were brought out through the incision. One milliliter of 10% sodium taurocholate (Sigma, lot 94F-5032) was manually infiltrated into the tail and body of the pancreas with a 25G needle. This method of pancreatitis induction is simple, predictable, and highly reproducible [ 1, 2, 61 and is therefore very suitable for NMR studies. Animals were reoperated at various time periods (between 20 and 360 min, see Table 1) at which time the pancreas was quickly removed and placed in chilled (0-4°C) phys-
iologic solution with oxygen bubbling in the NMR tube (10 mm diameter). The NMR spectrum of the whole pancreas was immediately recorded. Control rats were injected with saline solution into the pancreas. A total of 25 pancreases were examined (see Table 1). Preparation of extracts. In order to assign the 31P NMR signals, perchloric acid extracts of normal and sodium taurocholate-treated pancreases were made, as previously described [ 71. 3’P NMR studies. The NMR spectrometer was Bruker AM-360 WB operating at 145.78 MHz for “P measurements, equipped with a variable frequency IO-mm probe. The temperature was 2 f 1“C. The conditions of the NMR measurements of whole pancreases are described in the legend to Fig. 1. The 31P spectra of the extracts were recorded at various pH values. The titration
TABLE 1 THECORRELATIONSBETWEENTHETIMEELAPSEDFROMINDUCTIONOFPANCREATITIS,THENMR SPECTRALCHANGES,ANDTHEPATHOL~GICFINDINGS Interval from sodium taurocholate injection (mitt)
PH”
Normal (n = 4) Controld (n = 5) 20 25 30 30 30 45 45 60 75 100 120 135 140 140 180 360
7.20 + 0.06 7.20 + 0.06 7.09 7.08 6.92 7.24 7.08 7.24 7.08 7.09 7.19 7.02 7.11 7.22 7.30 7.11 7.17 7.13
PATP/Pi 0.7 2 0.2 0.7 + 0.2 0.14 0.07 0.09 0.2 0.21 0.49 0.12 0.13 0.13 0.05 0.14 0.05 0.09 0.09 0.15 0.08
Macroscopic damagec
Histologic damage ’
z ++ ++++ +++ +
z ++ ue up ue + Ue +++
!s ++ +++ +++ ++++ ++ ++++ ++++ ++++ ++ +++
LIPancreatic intracellular pH as determined by the chemical shift of inorganic phosphate. b The ratio between the intensity of the signals of ,!?ATP and inorganic phosphate. ‘The severity of the damage was graded from + to ++++. Izi = no damage. d 30, 50, 90, 120, and 160 min after saline injection. e u-Undetermined.
Ue
+++ Ue ++ ll= u’ ++++ Ue Lie
174
JOURNAL OF SURGICAL RESEARCH: VOL. 43, NO. 2, AUGUST
1987
the extension of necrotic and hemorrhagic areas and to the presence of saponification of fatty tissue within and around the pancreas. The histologic sections were reviewed by one of us (U.S.) without knowing the experimental conditions and the NMR spectrum of each pancreas. RESULTS
The NMR spectra of the pancreases were recorded at room temperature at the beginning of this study. The high energy phosphorous compounds, phosphocreatine (PCr) and adenosine triphosphate (ATP), are very labile under ischemic conditions, and our initial spectra contained virtually no PCr and minute amounts of ATP. Subsequent NMR studies were performed at low temperatures (2 f 1“C) with oxygen bubbling, resulting in 10.0 5.0 0.0 -10.0 -20.0 minimal PCr and ATP decomposition in the PPM NMR tube. FIG. 1. A series of j’P NMR spectra of whole rat panThe NMR spectrum of the intact normal creases before (a) and after (b-d) induction of pancreapancreas is shown in Fig. la. The signal at titis. = Interval after injection of sodium taurocholate, in minutes. Each spectrum is a collection of 400 scans 0.49 ppm was assigned as glycerophospho(duration 10 min) by applying 45” radio frequency choline (GPC) on the basis of its chemical pulses (13 psec) and relaxation delay of 1 sec. The spec- shift relative to external references and by its tra were collected with a 4K data points at a 7300-Hz (50 pH independence. GPC identification was ppm) spectral width. No proton decoupling was applied. verified by the addition of the genuine comFor detailed information regarding the assignment of pound to extracts where the NMR signal signals see text. coincided with the naturally occurring signal. All chemical shifts of phosphorous comcurves of their chemical shifts (6) were compounds in the NMR spectra of the pancreas puter fitted and the resultant values of acidic were calibrated using GPC as an internal 6, basic 6, and pK were compared with those standard at 0.49 ppm [9]. The signal of inorof known genuine compounds. Each spec- ganic phosphate (Pi) appeared in the region trum of the extract was a result of 600 scans 2.5-2.7 ppm; its exact chemical shift was of pancreatic inwith 45” pulses and relaxation delays of 1 used for the determination tracellular pH. Two peaks appeared in the sec. Proton decoupling was applied. Pathologic examination. After removal phosphomonoester region: a prominent one each pancreas was macroscopically examat about 4.2 ppm and another at about 3.7 ined for the presence of edema, hemorppm. These two peaks were identified by their chemical shifts and pK’s, as determined rhages, and necrotic areas. Following the NMR measurements the in extracts, as phosphoethanolamine (PEA) whole pancreas was fixed in buffered formaand phosphorylcholine (PCho), respectively. lin for 24 hr and embedded in paraffin. The signal of PCr (-2.5 ppm) was smaller Seven-micron-thick longitudinal sections of than the three signals of ATP (-5.1, - 10.0, the whole pancreas were stained with hema- 19.1 ppm) (see Discussion). The two peaks toxylin and eosine. The intensity of the le- at - 10.8 and - 12.3 ppm correspond to disions was evaluated and graded according to phosphodiester compounds, such as uridine I
t
KAPLAN
ET AL.: 3’P NMR OF PANCREATITIS
diphosphoglucose (UDPG). Control animals exhibited no changes in the spectra of pancreases removed 30, 50, 90, 120, and 160 min afier saline injection. Their spectra were practically identical to the spectrum of the normal pancreas shown in Fig. la. Sodium taurocholate-treated pancreases exhibited NMR spectral changes that became more pronounced as the disease progressed (Figs. lb, c, and d). The principal changes were gradual diminution of PCr and ATP and a concomitant increase of Pi. Thirty minutes after induction of pancreatitis there was only a slight decrease of PATP and a slight increase of Pi (Fig. lb). These changes were more pronounced 60 min after injection (Fig. lc), and virtually all high energy compounds disappeared after 140 min (Fig. Id). The NMR spectral changes correlated better with the macroscopic and microscopic features of the examined pancreas than with the time elapsed from sodium taurocholate injection. This was evident in several pancreases where minor spectral changes 2 and 3 hr after induction of pancreatitis were associated with minimal pathologic damage. It is noteworthy that while the PCr peak was initially smaller than ATP peaks, it did not completely disappear before ATP depleted (see Discussion). With the development of pancreatitis, a new peak appeared in the phosphodiester region, at -0.16 +. 0.04 ppm. This peak was found only in the early phases of pancreatitis and disappeared later when signs of severe damage to the organ predominated. It was found both in the whole organ and in extracts of pancreases treated with sodium taurocholate, indicating that the signal corresponds to a small phosphorus-containing molecule. The intracellular pH of the normal and control pancreases, as determined by the Pi chemical shifts, was in the range 7.14-7.26. In deseased pancreases only a slight pH decrease was found, in the range 6.92-7.30. However, pH changes were not progressive and did not parallel the progression of the disease.
175
No changes in the GPC signal were found in the early stages of pancreatitis. In the more severe stages some pancreases showed a marked depletion of GPC (Fig. Id), while others showed no change. Macroscopic appearance. Pancreases from control animals receiving saline injection showed no gross lesions, edema, or hemorrhages. The pancreas illustrated in Fig. lb appeared edematous with minimal hemorrhages. Extensive macroscopic damage was observed in the pancreas which had the most dramatic NMR spectral changes (Fig. Id). Histologic Jindings. The control pancreases showed a normal appearance of the lobules and surrounding fatty tissue (Fig. 2). The pathologic changes associated with mild NMR spectral changes consisted of few necrotic pancreatic lobules and mild saponification of the intralobular fatty tissue (Fig. 3). The severely affected animals (NMR spectrum-Fig. Id) showed nearly complete necrosis of the pancreatic parenchyma and severe fat necrosis in the intra- and peripancreatic fatty tissue (Fig. 4). Table 1 presents the correlations between the time elapsed from pancreatitis induction, the NMR spectral changes, and the pathologic lesions. The ratio of PATP to Pi is an index of the high energy compounds levels. The best correlation of the actual pathologic damage is with NMR spectral changes of these high energy compounds. DISCUSSION
The study described here points to the applicability of NMR spectroscopy for the quantitative measurements of metabolic changes that occur during acute pancreatitis and for the assessment of the severity of the disease. Such objective parameters are essential for both clinical evaluation and experimental research. Our data show a remarkable change in the “P NMR spectrum of the rat pancreas after the induction of pancreatitis, which correlated with the clinical course of the disease. The most significant changes were the disappearances of the high energy
176
JOURNAL OF SURGICAL
RESEARCH: VOL. 43, NO. 2, AUGUST
1987
FIG. 2. Saline injected control pancreas. Preservation of pancreatic glands and the islands of Langerham. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. 1a.)
compounds ATP and PCr. These spectral changes were found to be better indicators of the actual damage than the time elapsed from the sodium taurocholate injection.
The end result of the pathogenetic processes of acute pancreatitis is enzymatic destruction of pancreatic cells [lo]. Since high energy phosphorous compounds are essen-
FIG. 3. Mild necrotic changes. Few pancreatic lobules are necrotic. Peripancreatic fatty tissue shows fat necrosis. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. 1b.)
KAPLAN
ET AL.: “P NMR OF PANCREATITIS
FIG. 4. Severe necrotic changes. The pancreas is totally necrotic. Fatty tissue shows severe necrosis and saponification. Inflammatory cells around necrotic pancreas and fat. Hematoxylin eosine X 40. (The NMR spectrum of this pancreas is shown in Fig. Id.)
tial in preserving cell integrity, their depletion in the presence of pancreatic cell necrosis is expected. In the early phases of the disease only edema and minimal necrosis were found, and the NMR spectrum showed a minimal change. In the more severe stages the histologic picture showed extensive necrosis, and in the concomitant NMR spectra virtually all high energy compounds disappeared. These findings support the hypothesis of Becker et al. [3] that the progressive falls of ATP and PCr are signposts of the progression from edematous reversible to necrotizing irreversible pancreatitis. As shown in the present study, these signposts can be detected by 31P NMR. The pathologic damage in acute pancreatitis spreads from local necrosis in the early phases toward diffuse necrosis in the advanced states [8, lo]. This process was demonstrated in the present study by the fact that PCr did not completely disappear before ATP depleted, as would be expected on biochemical grounds if the whole organ was simultaneously damaged. There is a major ad-
vantage in measuring the whole intact organ in one examination, as is done by NMR, in comparison with techniques that require biopsy samples and measure only a limited portion of the organ. The 31P NMR spectrum of the normal pancreas is governed by signals in the phosphomonoester region, which were identified as PEA and PCho, and there is a high peak of GPC in the phosphodiester region. PEA showed no change during acute pancreatitis, while GPC was depleted in some severely damaged pancreases concomitant with a slight increase in PCho. No definitive conclusion can be drawn as these results were not reproducible. The new peak which appeared at about -0.16 ppm in the early stages of pancreatitis is not yet identified. The appearance of this peak both in whole organs and extracts indicates that it is a low-molecular-weight compound, presumably a degradation product of membranous phospholipids. Hypothesizing that it may be of diagnostic value as a marker, we are pursuing the assignment of
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the signal in the hope that it may contribute to understanding the pathogenesis of pancreatitis. Since our experiments were conducted under relatively ischemic conditions, the concentrations of PCr in our normal pancreas must have been lower than those in the live organ. This fact is of minor importance in the present study, since all the pancreases were examined under identical conditions, and conclusions were drawn from comparative rather than absolute values. This principle of comparative studies is also the basis for the validity of the histologic examinations. Moreover, as the pancreases were in chilled physiologic solution throughout NMR measurements, spontaneous autolysis was negligible. We are currently performing 3’P NMR studies of perfused rat pancreas in a study on the evolvement of various forms of pancreatitis [ 1, 11, 121 and on the efficacy of different therapeutic approaches. The results of the present study constitute, in our opinion, a firm basis for future clinical studies with the aid of topical NMR to evaluate the severity and prognosis of acute pancreatitis. REFERENCES 1. Aho, H. J., Nevalainen, T. J., and Aho, A. J. Experimental pancreatitis in the rat. Eur. Surg. Res. 15: 28, 1983.
1987
Bawnik, J. B., Orda, R., and Wiznitzer, T. Acute necrotizing pancreatitis: An experimental model. Dig. Dis. Sci. 19: 1143, 1974. 3. Becker, H., Vinten-Johansen, J., Buckberg, G. D., and Bugyi, H. I. Correlation of pancreatic blood flow and high energy phosphates during experimental pancreatitis. Eur. Surg. Res. 14: 203, 1982. 4. Cohen, J. S., Knop, R. H., Navon, G., and Foxall, D. Nuclear magnetic resonance in biology and medicine. Ll$ Chem. Rep. 1: 368, 1983. 5. Gadian, D. G. Nuclear Magnetic Resonance and Its Applications to Living Systems. New York: Oxford Univ. Press, 1982. Pp. l-70. 6. Hadas, N., Orda, R., Orda, S., Bawnik, J. B., and Wiznitzer, T. Experimental acute pancreatitis in rats caused by intraparenchymal injection of sodium taurocholate. Isv. J. Med. Sci. 19: 194, 1983. I. Lawry, 0. H., and Passoneau, J. V. A Flexible System of Enzymatic Analysis. New York: Academic Press, 1972. Pp. 120-124. 8. Longnecker, 0. S. Pathology and pathogenesis of diseases of the pancreas. Amer. J. Pathol. 107: 103, 1982. 9. Navon, G., Ogawa, S., Shulman, R. G., and Yamane, T. “P nuclear magnetic resonance studies of Ehrlich ascites Tumor cells. Proc. Natl. Acad. Sci. USA 14: 87, 1917. 10. Robbins, S. L., Cortan, R. S., and Kumar, V. (Eds.) Pathologic Basis of Disease. 3rd ed. Philadelphia: Saunders, 1984. Pp. 963-965. 11. Saharia, P., Margolis, S., Zuidema, G. D., and Cameron, J. L. Acute pancreatitis with hyperlipemia: Studies with an isolated perfused canine pancreas. Surgery 82: 60, 1977. 12. Sanfey, H., Bulkley, G. B., and Cameron, J. L. The pathogenesis of acute pancreatitis. Ann. Surg. 201: 633, 1985. 13. Silen, W., and Steer, W. L. Pancreas. In S. I. Schwartz (Ed.) Principles of Surgery, 4th ed. New York: McGraw-Hill, 1984. Pp. 1350-1353. 2.