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31P NMR studies of excised gray and white calf brain
Comp. Biochem. Physiol. Vol. 94B, No. 4, pp. 679-685, 1989 Printed in Great Britain
0305-0491/89 $3.00 + 0.00 © 1989 Pergamon Press pie
31p NMR STUDIES OF EXCISED GRAY A N D WHITE CALF BRAIN C. TYLER BURT,*t H.-M. CHENG~ and F. JUNGALWALLA§ *National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA (Tel: (919) 541-4535); ~Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, Massachusetts, USA; and §Shriver Center, Waltham, Massachusetts, USA
(Received 7 April 1989)
Abstract--1. 31p NMR examination of isolated calf gray and white matter reveals that white matter contains higher levels of the phosphodiester glycerolphosphoryl choline (GPC) than gray. 2. It is suggested that GPC may play a role in maintaining the level of phospholipids present by inhibition of phospholipases. 3. The spectra also reveal a skewed peak whose maximum is at - 11 ppm which is inferred to arise from myelin-like structures. 4. The results show that phosphorus spectra from the brain must be carefully considered whether they arise from the same type tissue or represent a mixed sample since variation in results may represent anatomy as well as physiology.
INTRODUCTION Biological N M R studies have grown over the past decade with the chief nucleus of observation tending to be phosphorus (3lp) (Gadian and Radda, 1981; Burt, 1982; Burt and Koutcher, 1984). One of their interesting outgrowths has been an appreciation of the role phosphodiesters play as markers and perhaps as modulators of tissue differentiation. By phosphodiesters we refer to the water-soluble metabolites that are observable by N M R and not the phospholipids where the phosphodiester linkage is generally N M R "invisible". They are the backbone of the phosphatidyl lipids such as glycerolphosphorylcholine (GPC) and glycerolphoryletbanolamine (GPE). As outlined by Burt and Ribolow (1984) they are often seen in concentrations in the m M level and vary substantially according to the level of differentiation in a wide variety of animal tissue. This study was specifically initiated to see if differences in the noncyclic phosphodiesters could be seen between gray and white matter in developing brain. The convoluted nature of brain makes interpretation of its spectra from whole tissue very difficult for N M R since N M R methods sample vols of at least 1 cm <3) in in vivo applications (Chance et al., 1985). The spatial resolution is also a problem for small animal organs like kidney that have macroscopic anatomic hetereogeneity on the m m level. The anatomic heterogeneity has parallel biochemical examples and one notes that as long as 30 years ago biochemical studies on kidney showed a distribution of phosphodiesters that was extremely asymmetric from cortex to medulla (Schimassek et al., 1959). They showed this heterogeneity by dissecting the
kidney into the appropriate fractions and doing biochemical analysis. Based on this and similar experiments, we decided the easiest and probably best strategy to test for hetereogeneity in brain was also to first dissect out gray and white matter and examine the isolated sample. This of course precludes most energetic experiments but allows an inventory to be made of compounds present. MATERIALS AND METHODS
Biological materials Calf brains from dairy cattle (Bos taurus) were obtained from a local abattoir and transported on ice to the experimental site at Massachusetts Institute of Technology. Gray and white matter were separated by dissecting the tissue using its visual characteristics. The separated samples were placed in 10 mm NMR tubes together with enough D20 to allow the machine to be shimmed. The NMR runs were performed at ambient temperature (22-25°C). Perchloric acid extracts were made by homogenizing brain or isolated fractions, such as white matter (~5-10g), with 5 vols of 5% perchloric acid after which conventional purification methods were used (Barany and Glonek, 1982). NMR measurements Except for T 1measurements, a modified Bruker 270 MHz ~H, 109.3 MHz 3tp spectrometer was used togther with 10 mm diameter NMR tubes. Typical conditions were 45 ° pulse angle, 3000 Hz sweepwidth, proton coupled, one second repetition rate and 4000 sampling points. Brain samples needed only 5-10 min of signal averaging to obtain interpretable signals although longer collection periods were used. The spectrometer was sufficiently stable so that chemical shifts could be determined by first running a sample of 20 mM phosphocreatine (PCr) in 0.1 M KC1 and setting its chemical shift to -3.1 ppm. All subsequent spectra are referenced to this peak. The value of -3.1 ppm was selected since it makes the data in this work comparable to previous ones (Burtet al., 1977a). Longitudinal relaxation time (Tt) measurements were performed on a Nicolet 360 MHz spectrometer at the National Institute of Environmental Health Sciences (NIEHS).
tAuthor to whom all correspondence should be addressed at: NIEHS, LMB, P.O. Box 12233, MD 17-05, Research Triangle Park, NC, 27709, USA (Tel: 919-541-4535). 679
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31p NMR S p e c t r u m of Myelin Fraction
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Fig. 1. (A) 31p N M R spectra of intact white brain. In this and all succeeding spectra, resonances to the right are upfield. The large sharp peak is inorganic phosphate (P~). The peak for glycerol phosphorylcholine is seen as the shoulder on the right-hand side of Pi. The peak is stable with time and further, does not change its resonance position if tissue pH was adjusted by soaking in alkaline buffer. The internal pH did change as can be noted by the shift in the inorganic Pi to a more downfield position. Gray matter did not show a corresponding prominent resonance where GPC is seen in this figure. The broad asymmetric resonance centered at - 11 ppm is also much reduced in gray matter (see Fig. 2). The inset shows an expansion of Pi and GPC region showing more clearly the appearance of GPC and its invariance with time. In addition, the 6-8 hr average in which the sample was soaked with alkaline (pH 10) buffer resulted in a pH shift of the P~ peak but no change in GPC. Here the presence of GPE can be seen as the upfield shoulder on the Pi resonance. Pulse conditions as listed in text and time of accumulation shown on figure. (B) 3~p N M R spectrum of a perchloric acid (PCA) extract of calf brain white matter. The presence of both GPC and GPE can be clearly discerned. The 31p N M R extract results therefore confirm the results seen in Fig. 1 in intact tissue. One notes the large resonance which is at - 11 ppm upfield of GPC is missing in the extract. (C) 3tp N M R spectrum of a myelin fraction from calf white matter. Its main feature is the broad assymmetric peak at - 11 ppm. This is what is expected from phospholipids in a bilayer vesicle. The sweep width for this spectrum is 4000 Hz. The spectra in (A)-(C) are all scaled differently so no uniform ppm scale is included. All peaks and ppm values were calculated as described in the text. This field strength is only about 30% greater than 270 MHz and so relaxation times should be quite comparable. Adult cow brain was obtained from a slaughter house approximately 1 hr driving distance from NIEHS and samples were
separated into gray and white matter using visual appearance as described above. The animals were at least 1 yr old. A 90 ° pulse (39 #sec) was previously determined and the pulse program used was the Nicolet T I l R with a 10 sec
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Phosphodiester distribution in the brain repetition rate and 200 accumulations. Values of phosphate concentrations are listed as % of observable phosphate.
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INTACT WHITEMATTER 81p NMR
RESULTS Figure 1A shows a typical spectrum of isolated white matter. Besides the large P~ peak, a peak at approximately - 0 . 1 ppm is clearly visible. The peak is due to GPC. That the peak is G P C is inferred from the chemical shift which, in general, is unique for a particular phosphodiester, but also the fact that the resonance position does not change if tissue p H is changed by soaking the brain in alkaline buffer (Burt et al., 1977b). Before running the N M R spectra, the PCr and A T P had broken down due to the method of sample collection and transportation. In results on rat brain, which are removed and then immediately frozen, we and others find PCr and A T P have been broken down to N M R unobservable levels in less than 15 mins after dissection (Chance et al., 1979). This means that any conventional method of isolation short of funnel freezing will have little A T P or PCr present. However, phosphodiesters in isolated tissue are generally very stable over 1-2 hr so that their level would not be expected to change in the manner of PCr and A T P (Dawson et al., 1980; Rhodes et al., 1983). Figure 1 shows that over an 8 hr period, G P C in brain samples is stable in the N M R tube. Figure 2 shows a spectra of gray matter corresponding to Fig. 1A. The pH in the sample is more basic than in white matter so that the appearance of G P C would not be obscured by the inorganic phosphate (P~) peak. The low level of G P C seen in the figure is therefore real. Figure 3 shows a comparison of both gray and white matter together. The difference in p H as calculated using the chemical shift difference between G P C and Pi was seen in all samples with the p H of gray matter being 6.5 (three samples) and that of white matter 6.1 (three samples). The reason for this difference is not known but may be related to differences in buffering power of the respective tissues. Previous reports from rabbits observed that lactate levels are higher in gray than white matter, so this source of p H differential seems ruled out since higher lactate would imply a lower p H
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Fig. 3. Comparison spectra of intact gray and white matter. The samples are both from the same calf and is different from those shown in Figs 1 and 2. There were 200 accumulations for both samples with other pulse conditions as listed in the text. (Petroff et aL, 1987). It should be noted that different p H ' s have been noted between p H predicted by inorganic phosphate and phosphorylethanolamine (Corbett et al., 1987). F r o m such spectra it is clear that the level of G P C is high in white matter. Table 1 lists an average value for relative G P C concentration for intact gray and white matter in calf brain. It can be seen that G P C is at least 3-fold more concentrated in white matter than gray. Absolute concentrations are not possible due to the presence of D 2 0 in part of the sample space. However, with a few assumptions one can calculate a m M (/z m / g wet wt) concentration and this is given in parenthesis in the table. In addition one has the problem of partial saturation as referred to in Table 1. We have measured the T~'s of both G P C and inorganic phosphate in adult gray and white matter. The values for TI were 2.2 sec for G P C in gray; 1.9 sec for G P C in white; Pi in gray is 1.5 sec and Pi in white is 2.0 sec. The simplest equation to describe the relative saturation is Iexp = I0(1 -- e-~/r~), Table 1. Phosphate type in phosphate of total NMR observable phosphate % Phosphate*
'
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'
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-1 -15-
pprn
Fig. 2. NMR spectrum of intact gray matter. Conditions and assignments are as in Fig. I(A). The sample was more alkaline than for Fig. I(A) so that any GPC present would not be obscured by acidic shift of the Pi peak. There were 200 accumulations with other pulse conditions as listed in the text.
Tissue type PME Pi GPC Gray (5)t 28~ _+2§ 70 + 2 2 + 2 (0.7)11 White (5) 22 + 2 72 + 3 7¶ _+2 (4) *Phosphate areas were determined by cutting and weighting. The broad phosphate peak was averaged as part of the baseline. If that peak was cut out it represented 64 and 77% of totally observable phosphate in gray and white matter, respectively. tThe number of determinations was five, ~:Actualconcentration willdepend on relaxation times and, based on our relaxation times, this should not be a problem. §Values are standard deviation. IIValuein parenthesis is calculated in mM. This was done using total phosphate, phospholipid and water content values derived from various Handbooks (Altman and Dittmar, 1974;Speetor, 1956). One calculates a total observable phosphate concentration of 30 mM (#m/g wet wt) in gray and 40 in white matter. ¶Significantly higher than the corresponding tissue at the 1% level using the t-test.
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where Iexp is the experimental magnetization intensity, I0 the equilibrium magnetization, z the experimental repeat time and T l the relaxation time. The relative saturations would be 0.37, 0.41, 0.49 and 0.39 using the Tl's above. Thus, Pi in gray matter may be slightly overcounted. All the other values appear equivalent. This means all signals should be similarly saturated so that relative values should be subject to little distortion. A recent study shows a general increase in T~ for inorganic phosphate with development in an in vivo rat system (Stolk et al., 1987). However, the usual problem of differentiating brain from muscle, much less gray from white matter, makes any generalization from this study unclear. Even if the absolute T~'s are longer in calf brain than adult, our demonstration of little difference between gray and white matter should stand. The problem of tissue differentiation meant that only white matter could be isolated in a pure form. To aid in identification of compounds and to help clarify the nature of the broad peak in white matter, PCA extracts were made of it. The N M R spectrum of the perchloric extract of the white matter is shown in Fig. lB. GPC is readily seen and if one calculates the % of total phosphate the peak represents one obtains 6% of total phosphate which agrees well with the 7% value in Table 1. In addition to the peak due to GPC, the spectrum shows the presence of GPE. The latter peak was hidden in the intact tissue by the acidic state of the samples. From the literature it is known that GPE should be present in brain (Ansell and Norman, 1953; Altman and Dittmer, 1974). It is also interesting to note that there is a broad asymmetric hump present mainly in white matter with a maximum at roughly - 1 1 ppm which is not present in the extract. The peak's shape suggests the resonance probably results from organelle bilayer lipids which has been previously reported for mitochondria and synaptosomes (de Kruijff et al., 1982; Fuldner and Stadler, 1982). To investigate this point an N M R spectrum (Fig. 1C) of a myelin preparation was determined. The myelin fraction shows the characteristic pattern as seen in intact white matter (Fig. IA). It is possible that 31p N M R can be used to monitor myelin or vesicle formation in intact samples. Our results agree with those of Chang et al., (1985) although they only did whole brain observation and did not show the peak results mainly from white matter. This also serves as an auxiliary demonstration that the separation of gray and white matter has been successful since the white matter contains high levels myelin compared to gray (see Figs 1-3). In vivo spectra of brain taken with surface coils show a large resonance at approximately 0 p p m which was assigned to phospholipids (Cerdan et al., 1986). The same kind of results have also been reported in isolated cancer cells (Guidoni et aL, 1987). The considerations listed below apply to both systems: namely that st~ch phospholipids must not be in the form of bilayers as seen in the myelin spectra but rather packaged as the lipid in circulating phospholipids (HDLs, LDLs, etc.) which show a relatively sharp peak centered at approximately 0 ppm (Henderson et al., 1975). Also in this region are the resonances of the high energy carboxyl phosphates
such as phosphoenolpyruvate (Burt et al., 1979). Certainly our intact brain sample does not show as strong a resonance as seen in in vivo human, rabbit and dog brains collected at field strength below 2 Tesla. Thus, if such compounds are present in the calf they are labile and break down within the 0.5-1 hr that the sample transportation requires (Delpy et al., 1982). The situation has become even more complex with the recent observation that the intensity of the peak in the phosphodiester region varies in in vivo preparation according to field strength (Sauter and Ruden, 1987). Higher field strengths do not show nearly as large an amplitude as that at lower fields. Since our measurements were made at a field strength comparable to the high field of Sauter and Rudan, an alternative explanation for the lower total intensity in the phosphodiester region could be due just to field strength. At any rate it is apparent that the nature of the total observed in vivo intensity in the phosphodiester region is a difficult and evolving question; however, we show here that the use of intact separated samples allows a simplification that makes analysis of such questions easier. Examination of the spectra show the presence of resonances in the monoester region. Comparison of published chemical shifts and extract work show that when two peaks are present they are phosphorylcholine (PCh) and phosphorylethanolamine (PEth). Our results confirm a published paper by Gyulai et al. (1984), that PEth is present in newborn dog brain. It also confirms previous classical biochemical results. The presence of more phosphomonoesters in gray matter than white could provide extra acidic buffering capacity that would go along with the suggestion for the observation that gray matter reaches a less acidic final pH than white (see Table 1). DISCUSSION TO our knowledge, this represents the first attempt to characterize by 31p NMR, gray and white matter. The results have appeared earlier in abstract form (Burt et al., 1986), and an even more recent abstract confirms in elderly humans the results given here: namely, phosphodiesters are higher in white matter than gray and vice versa for phosphomonoesters (Smith et al., 1988). Subsequent reports by others have featured proton N M R results of differences between gray and white matter (Petroff et al., 1987). In the latter report most of the metabolites observed seem to undergo modest temporal changes compared to phosphocreatine and ATP. This is in keeping with our observation that GPC is not substantially metabolized. Other factors which might effect the results have been enumerated. We have found therefore, that in calf brain, phosphodiesters are high in white matter. It has been noted in almost all neonates that PEth is high and then decreases with age. But in comparison to this study one cannot judge if this is due to a uniform decrease in PEth or to some kind of tissue differentiation. The method presented here should allow such questions to be addressed. We have not investigated whether our change persists with time, however, studies in two other species seem to indicate that it is
Phosphodiester distribution in the brain
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Table 2. Some phospholipid % concentration in differentiating systems State of PL PDE System differentiation % PL* content conc:~ Reference Muscle White Rabbit Extensor Digitorum Longus 90 Burt et al. (1982) Sarcoplasmic Reticulum Guinea Pig Vastus 64 13.5 Sarzala et al. (1982) Red Rabbit Soleus 70 + Butt et al. (1982) Sarcoplasmic Reticulum Guinea Pig Soleus 43 9.5 + Sarzala et al. (1982) Brain Gray (human) 72§ 37 ? Spector (1956) White (human) 51§ 85 ? Spector (1956) Astrocytomas (human-all types) 6211 15 ? Yates et al. (1979) Glioblastoma 5011 14 ? Yates et al. (1979) *Percent phospholipid expressed as (mg of phospholipid/g tissue divided by mg of total lipid per gram tissue) x 100. tWhere known phospholipid content expressed as mg/g tissue. :[:Phosphodiesterconcentration (PDE) graded on a relative scale from absent ( - ) to present ( + ). ? mark means data is not present. §Values represent midrange values from Spector's (1956) handbook. 11Basedon the % P and on an average mol wt calculated for a fixed fatty acid composition and the percent distribution of headgroups listed in Yates et al. (1979). a uniform phenomenon that persists into adulthood. Pettegrew has shown in whole rat brain extract that the phosphodiester to total phosphate ratio increases past birth and then remains high (Pettegrew e t al., 1986a). His results in terms of adult rats agree with those of Glonek e t al. (1982). In human surface coil studies it also appears that the phosphodiester region seems more prominent in adults than children (Bottomley e t al., 1984; Z i m m e r m a n et al., 1985). The human studies, of course, have the problem of field strength dependent phenomena mentioned previously. Nevertheless, for both cases an increase in phosphodiesters due to white matter proliferation is consistent with the data. Besides the N M R results from brain, it is also known that several other organs have phosphodiesters distributed in a very asymmetric manner. Thus G P C is high in kidney medulla but low in the cortex (Schimassek e t al., 1959). Phosphodiesters are more prominent in red muscle than white (Burt et al., 1982), and in surface coil studies are seen to increase disproportionately in Duchenne Dystrophy (Youkin et al., 1987). Table 2 tabulates these differences and also lists some of the lipid differences between differentiated tissues. In addition, our finding has direct relevance for studies using P C A extracts of brains. Any conclusion drawn about variations in phosphodiester concentration as characteristic of a condition or disease must bear in mind that different cerebral tissue may vary both with respect to percentage of gray and white matter or differentiation. In general, the larger the specimen being examined, the larger the artifacts could be, since it will contain a larger mixture of gray and white matter. H o w this can be applied to a specific problem is seen in studies on extracts of brains subject to dementia (Barany e t al., 1985; Miatto e t al., 1986). They found elevated G P C levels in pathological extracts, however, they do not account for possible variations in gray and white matter ratios. Since some studies show there is more atrophy of gray matter than white in aging, the expected relative increase in white matter together with the present study could
explain the elevated G P C levels on anatomical rather than physiological grounds, particularly if controls are not good age matches for the dementia cases (Duara et al., 1986). In terms of the biochemical significance of the appearance of GPC, it must be related to phospholipid metabolism. It has been suggested that phosphodiesters are mainly an index of catabolism (Pettegrew e t al., 1986b). In this regard one notes that total phospholipid concentration increases dramatically in white matter. F o r example, in the human, phospholipid concentration per gram of tissue is 85 m g / g m in white matter vs 37 in gray (Spector, 1956). So one could argue higher G P C levels could simply be due to more substrate. However, this is probably too simple a view for as pointed out by Ribolow and Burt (1987), G P C can serve as a lysophospholipase inhibitor. This means PP COl=Ch__
Fig. 4. Redrawn general scheme showing how the processing of phospholipids can be seen as a cycle. PC and LPC are phosphatidylcholine and lysophosphatidylcholine and are membrane-bound. The other -glycerol phosphoryleholine (GPC), ~-glycerol phosphate (ctGP), choline (Ch), phosphocholine (PCh), cytidine triphosphate (CTP), pyrophosphate (PP) and cytidine diphosphocholine (CDPCh) are all water soluble. The enzymes involved are: (I) phospholipase; (2) lysolecithin acyl transferase; (3) lysoelcithinase; (4) phosphodiesterase; (5) choline kinase; (6) phosphocholine-CMP transferase; (7) CDP choline transferase; and (8) phospholipase C. The reported inhibition of reaction 3 by GPC is shown by a dotted line from GPC. Other conceivable inhibitions by PCh (or PEth) are also marked.
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G P C may be helping to maintain high phospholipid levels by decreasing the rate of flow of phospholipid through lysophospholipid hydrolysis. Indeed from Table 2 it can be seen that regardless of total phospholipid concentration, phosphodiesters such as G P C are high in the differentiated tissue which has the lower % phospholipid composition. The scheme suggested above can be viewed in terms of Fig. 4 where the lipid soluble metabolites are seen to be connected in a cycle to the water soluble ones. It is suggested that control of the water-soluble G P C both by generation through lipases and breakdown through diesterases can control lipid-related events via feedback inhibition. Ribolow and Burt (1987) demonstrate this inhibition in sperm and seminal plasma with G P C at the 1 m M level. To summarize then, our research points out there can be substantial differences in the phosphorus spectra of gray and white matter at a particular stage of development in calf brain. It further adds brain to the list of tissues in which phosphodiesters serve as potential markers and modulators of development to brain. REFERENCES
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Phosphodiester distribution in the brain Pettegrew J. W., Withers G., Panachalingam K. and Post J. F. M. (1986b) 31p Nuclear magnetic resonance spectroscopy of brain in aging and Alzheimer's disease. Proc. Fourth Meeting Int. Study Group on the Pharm. o f Memory Disorders Associated with Aging, pp. 57~58. Rhodes R, S., Jentoft J. E. and Robinson R. V. (1983) 3tp N M R studies of energy metabolism in perfused rat kidney. J. Surg. Rsch. 35, 373-382. Ribolow H. and Butt C. T. (1987) Analysis of lipid metabolism in semen and its implications. In Magnetic Resonance o f the Reproductive System (Edited by McCarthy S. O. and Hazeltine F.), SLACK Incorporated, New Jersey, pp. 128-136. Sarzala M. G., Szymanska G., Wiehrer W. and Pette D. 0982) Effects of chronic stimulation at low frequency of the lipid phase of sarcoplasmic reticuluum in rabbit fast-twitch muscle. Eur. J. Biochem. 123, 241. Sauter A. and Ruden M. (1987) Effects of calcium antagonists on high-energy phosphates in ischemic rat brain measured by alp NMR spectroscopy. Soc. Mag. Reson. Med. 4, 1-8. Schimassek H., Kohl D. and Butcher T. 0959) Glycerolphosphorylcholine, die Nierensulstanz Ma-Mark von Ullrich. Biochem. Zs. 331, 87-97.
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Smith C. D., Gallenstein L. G. III and Markesberry W. R. (1988) 31p spectroscopy in Alzheimer's and Pick's Disease using Post-Mortem Perchloric Acid Brain Extracts. Abstracts 7th A. Soc. Mag. Res. Meet. p. 44. Spector W. S. (1956) Handbook o f Biological Data, p. 70. W. B. Saunders Co., Philadelphia. Stolk J. A., Olsen J. L., Alderman D. W. and Schweizer M. P. (1987) Effects of age on apparent 3~p spin-lattice relaxation times of rat brain phosphates. Mag. Res. in Med. 5, 78. Yates A. J., Thompson D. K., Boesel C. P., Albrightson C. and Hart R. W. (1979) Lipid compositiQn of human neural tumors. J. Lipid Res. 20, 428. Youkin D. P., Berman P., Sladky J., Chee C., Bank W. and Chance B. (1987) 3tp N M R studies in Duchenne muscular dystrophy: Age-related metabolic changes. Neurology, Minneap., 37, 165-169. Zimmerman R. A., Bottomley P. A., Edelstein W. A., Hart H. R., R~iington R. N., Bilaniuk L. T., Grossman R. I., Goldberg H. I., Bruno L. and Kressel H. (1985) Proton imaging and phosphorus spectroscopy in a malignant gleoma. Am. J. Neuro. Radio. 6, 109-110.
0305-0491/89 $3.00 + 0.00 © 1989 Pergamon Press pie
31p NMR STUDIES OF EXCISED GRAY A N D WHITE CALF BRAIN C. TYLER BURT,*t H.-M. CHENG~ and F. JUNGALWALLA§ *National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA (Tel: (919) 541-4535); ~Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, Massachusetts, USA; and §Shriver Center, Waltham, Massachusetts, USA
(Received 7 April 1989)
Abstract--1. 31p NMR examination of isolated calf gray and white matter reveals that white matter contains higher levels of the phosphodiester glycerolphosphoryl choline (GPC) than gray. 2. It is suggested that GPC may play a role in maintaining the level of phospholipids present by inhibition of phospholipases. 3. The spectra also reveal a skewed peak whose maximum is at - 11 ppm which is inferred to arise from myelin-like structures. 4. The results show that phosphorus spectra from the brain must be carefully considered whether they arise from the same type tissue or represent a mixed sample since variation in results may represent anatomy as well as physiology.
INTRODUCTION Biological N M R studies have grown over the past decade with the chief nucleus of observation tending to be phosphorus (3lp) (Gadian and Radda, 1981; Burt, 1982; Burt and Koutcher, 1984). One of their interesting outgrowths has been an appreciation of the role phosphodiesters play as markers and perhaps as modulators of tissue differentiation. By phosphodiesters we refer to the water-soluble metabolites that are observable by N M R and not the phospholipids where the phosphodiester linkage is generally N M R "invisible". They are the backbone of the phosphatidyl lipids such as glycerolphosphorylcholine (GPC) and glycerolphoryletbanolamine (GPE). As outlined by Burt and Ribolow (1984) they are often seen in concentrations in the m M level and vary substantially according to the level of differentiation in a wide variety of animal tissue. This study was specifically initiated to see if differences in the noncyclic phosphodiesters could be seen between gray and white matter in developing brain. The convoluted nature of brain makes interpretation of its spectra from whole tissue very difficult for N M R since N M R methods sample vols of at least 1 cm <3) in in vivo applications (Chance et al., 1985). The spatial resolution is also a problem for small animal organs like kidney that have macroscopic anatomic hetereogeneity on the m m level. The anatomic heterogeneity has parallel biochemical examples and one notes that as long as 30 years ago biochemical studies on kidney showed a distribution of phosphodiesters that was extremely asymmetric from cortex to medulla (Schimassek et al., 1959). They showed this heterogeneity by dissecting the
kidney into the appropriate fractions and doing biochemical analysis. Based on this and similar experiments, we decided the easiest and probably best strategy to test for hetereogeneity in brain was also to first dissect out gray and white matter and examine the isolated sample. This of course precludes most energetic experiments but allows an inventory to be made of compounds present. MATERIALS AND METHODS
Biological materials Calf brains from dairy cattle (Bos taurus) were obtained from a local abattoir and transported on ice to the experimental site at Massachusetts Institute of Technology. Gray and white matter were separated by dissecting the tissue using its visual characteristics. The separated samples were placed in 10 mm NMR tubes together with enough D20 to allow the machine to be shimmed. The NMR runs were performed at ambient temperature (22-25°C). Perchloric acid extracts were made by homogenizing brain or isolated fractions, such as white matter (~5-10g), with 5 vols of 5% perchloric acid after which conventional purification methods were used (Barany and Glonek, 1982). NMR measurements Except for T 1measurements, a modified Bruker 270 MHz ~H, 109.3 MHz 3tp spectrometer was used togther with 10 mm diameter NMR tubes. Typical conditions were 45 ° pulse angle, 3000 Hz sweepwidth, proton coupled, one second repetition rate and 4000 sampling points. Brain samples needed only 5-10 min of signal averaging to obtain interpretable signals although longer collection periods were used. The spectrometer was sufficiently stable so that chemical shifts could be determined by first running a sample of 20 mM phosphocreatine (PCr) in 0.1 M KC1 and setting its chemical shift to -3.1 ppm. All subsequent spectra are referenced to this peak. The value of -3.1 ppm was selected since it makes the data in this work comparable to previous ones (Burtet al., 1977a). Longitudinal relaxation time (Tt) measurements were performed on a Nicolet 360 MHz spectrometer at the National Institute of Environmental Health Sciences (NIEHS).
tAuthor to whom all correspondence should be addressed at: NIEHS, LMB, P.O. Box 12233, MD 17-05, Research Triangle Park, NC, 27709, USA (Tel: 919-541-4535). 679
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31p NMR S p e c t r u m of Intact Calf White M a t t e r
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SIp NMR S p e c t r u m of PCA E x t r a c t
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31p NMR S p e c t r u m of Myelin Fraction
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Fig. 1. (A) 31p N M R spectra of intact white brain. In this and all succeeding spectra, resonances to the right are upfield. The large sharp peak is inorganic phosphate (P~). The peak for glycerol phosphorylcholine is seen as the shoulder on the right-hand side of Pi. The peak is stable with time and further, does not change its resonance position if tissue pH was adjusted by soaking in alkaline buffer. The internal pH did change as can be noted by the shift in the inorganic Pi to a more downfield position. Gray matter did not show a corresponding prominent resonance where GPC is seen in this figure. The broad asymmetric resonance centered at - 11 ppm is also much reduced in gray matter (see Fig. 2). The inset shows an expansion of Pi and GPC region showing more clearly the appearance of GPC and its invariance with time. In addition, the 6-8 hr average in which the sample was soaked with alkaline (pH 10) buffer resulted in a pH shift of the P~ peak but no change in GPC. Here the presence of GPE can be seen as the upfield shoulder on the Pi resonance. Pulse conditions as listed in text and time of accumulation shown on figure. (B) 3~p N M R spectrum of a perchloric acid (PCA) extract of calf brain white matter. The presence of both GPC and GPE can be clearly discerned. The 31p N M R extract results therefore confirm the results seen in Fig. 1 in intact tissue. One notes the large resonance which is at - 11 ppm upfield of GPC is missing in the extract. (C) 3tp N M R spectrum of a myelin fraction from calf white matter. Its main feature is the broad assymmetric peak at - 11 ppm. This is what is expected from phospholipids in a bilayer vesicle. The sweep width for this spectrum is 4000 Hz. The spectra in (A)-(C) are all scaled differently so no uniform ppm scale is included. All peaks and ppm values were calculated as described in the text. This field strength is only about 30% greater than 270 MHz and so relaxation times should be quite comparable. Adult cow brain was obtained from a slaughter house approximately 1 hr driving distance from NIEHS and samples were
separated into gray and white matter using visual appearance as described above. The animals were at least 1 yr old. A 90 ° pulse (39 #sec) was previously determined and the pulse program used was the Nicolet T I l R with a 10 sec
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Phosphodiester distribution in the brain repetition rate and 200 accumulations. Values of phosphate concentrations are listed as % of observable phosphate.
PI~ i
INTACT WHITEMATTER 81p NMR
RESULTS Figure 1A shows a typical spectrum of isolated white matter. Besides the large P~ peak, a peak at approximately - 0 . 1 ppm is clearly visible. The peak is due to GPC. That the peak is G P C is inferred from the chemical shift which, in general, is unique for a particular phosphodiester, but also the fact that the resonance position does not change if tissue p H is changed by soaking the brain in alkaline buffer (Burt et al., 1977b). Before running the N M R spectra, the PCr and A T P had broken down due to the method of sample collection and transportation. In results on rat brain, which are removed and then immediately frozen, we and others find PCr and A T P have been broken down to N M R unobservable levels in less than 15 mins after dissection (Chance et al., 1979). This means that any conventional method of isolation short of funnel freezing will have little A T P or PCr present. However, phosphodiesters in isolated tissue are generally very stable over 1-2 hr so that their level would not be expected to change in the manner of PCr and A T P (Dawson et al., 1980; Rhodes et al., 1983). Figure 1 shows that over an 8 hr period, G P C in brain samples is stable in the N M R tube. Figure 2 shows a spectra of gray matter corresponding to Fig. 1A. The pH in the sample is more basic than in white matter so that the appearance of G P C would not be obscured by the inorganic phosphate (P~) peak. The low level of G P C seen in the figure is therefore real. Figure 3 shows a comparison of both gray and white matter together. The difference in p H as calculated using the chemical shift difference between G P C and Pi was seen in all samples with the p H of gray matter being 6.5 (three samples) and that of white matter 6.1 (three samples). The reason for this difference is not known but may be related to differences in buffering power of the respective tissues. Previous reports from rabbits observed that lactate levels are higher in gray than white matter, so this source of p H differential seems ruled out since higher lactate would imply a lower p H
J
INTACTGREYMATTER
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;
0
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Fig. 3. Comparison spectra of intact gray and white matter. The samples are both from the same calf and is different from those shown in Figs 1 and 2. There were 200 accumulations for both samples with other pulse conditions as listed in the text. (Petroff et aL, 1987). It should be noted that different p H ' s have been noted between p H predicted by inorganic phosphate and phosphorylethanolamine (Corbett et al., 1987). F r o m such spectra it is clear that the level of G P C is high in white matter. Table 1 lists an average value for relative G P C concentration for intact gray and white matter in calf brain. It can be seen that G P C is at least 3-fold more concentrated in white matter than gray. Absolute concentrations are not possible due to the presence of D 2 0 in part of the sample space. However, with a few assumptions one can calculate a m M (/z m / g wet wt) concentration and this is given in parenthesis in the table. In addition one has the problem of partial saturation as referred to in Table 1. We have measured the T~'s of both G P C and inorganic phosphate in adult gray and white matter. The values for TI were 2.2 sec for G P C in gray; 1.9 sec for G P C in white; Pi in gray is 1.5 sec and Pi in white is 2.0 sec. The simplest equation to describe the relative saturation is Iexp = I0(1 -- e-~/r~), Table 1. Phosphate type in phosphate of total NMR observable phosphate % Phosphate*
'
'
'
10 5 O -
b'g
-1 -15-
pprn
Fig. 2. NMR spectrum of intact gray matter. Conditions and assignments are as in Fig. I(A). The sample was more alkaline than for Fig. I(A) so that any GPC present would not be obscured by acidic shift of the Pi peak. There were 200 accumulations with other pulse conditions as listed in the text.
Tissue type PME Pi GPC Gray (5)t 28~ _+2§ 70 + 2 2 + 2 (0.7)11 White (5) 22 + 2 72 + 3 7¶ _+2 (4) *Phosphate areas were determined by cutting and weighting. The broad phosphate peak was averaged as part of the baseline. If that peak was cut out it represented 64 and 77% of totally observable phosphate in gray and white matter, respectively. tThe number of determinations was five, ~:Actualconcentration willdepend on relaxation times and, based on our relaxation times, this should not be a problem. §Values are standard deviation. IIValuein parenthesis is calculated in mM. This was done using total phosphate, phospholipid and water content values derived from various Handbooks (Altman and Dittmar, 1974;Speetor, 1956). One calculates a total observable phosphate concentration of 30 mM (#m/g wet wt) in gray and 40 in white matter. ¶Significantly higher than the corresponding tissue at the 1% level using the t-test.
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where Iexp is the experimental magnetization intensity, I0 the equilibrium magnetization, z the experimental repeat time and T l the relaxation time. The relative saturations would be 0.37, 0.41, 0.49 and 0.39 using the Tl's above. Thus, Pi in gray matter may be slightly overcounted. All the other values appear equivalent. This means all signals should be similarly saturated so that relative values should be subject to little distortion. A recent study shows a general increase in T~ for inorganic phosphate with development in an in vivo rat system (Stolk et al., 1987). However, the usual problem of differentiating brain from muscle, much less gray from white matter, makes any generalization from this study unclear. Even if the absolute T~'s are longer in calf brain than adult, our demonstration of little difference between gray and white matter should stand. The problem of tissue differentiation meant that only white matter could be isolated in a pure form. To aid in identification of compounds and to help clarify the nature of the broad peak in white matter, PCA extracts were made of it. The N M R spectrum of the perchloric extract of the white matter is shown in Fig. lB. GPC is readily seen and if one calculates the % of total phosphate the peak represents one obtains 6% of total phosphate which agrees well with the 7% value in Table 1. In addition to the peak due to GPC, the spectrum shows the presence of GPE. The latter peak was hidden in the intact tissue by the acidic state of the samples. From the literature it is known that GPE should be present in brain (Ansell and Norman, 1953; Altman and Dittmer, 1974). It is also interesting to note that there is a broad asymmetric hump present mainly in white matter with a maximum at roughly - 1 1 ppm which is not present in the extract. The peak's shape suggests the resonance probably results from organelle bilayer lipids which has been previously reported for mitochondria and synaptosomes (de Kruijff et al., 1982; Fuldner and Stadler, 1982). To investigate this point an N M R spectrum (Fig. 1C) of a myelin preparation was determined. The myelin fraction shows the characteristic pattern as seen in intact white matter (Fig. IA). It is possible that 31p N M R can be used to monitor myelin or vesicle formation in intact samples. Our results agree with those of Chang et al., (1985) although they only did whole brain observation and did not show the peak results mainly from white matter. This also serves as an auxiliary demonstration that the separation of gray and white matter has been successful since the white matter contains high levels myelin compared to gray (see Figs 1-3). In vivo spectra of brain taken with surface coils show a large resonance at approximately 0 p p m which was assigned to phospholipids (Cerdan et al., 1986). The same kind of results have also been reported in isolated cancer cells (Guidoni et aL, 1987). The considerations listed below apply to both systems: namely that st~ch phospholipids must not be in the form of bilayers as seen in the myelin spectra but rather packaged as the lipid in circulating phospholipids (HDLs, LDLs, etc.) which show a relatively sharp peak centered at approximately 0 ppm (Henderson et al., 1975). Also in this region are the resonances of the high energy carboxyl phosphates
such as phosphoenolpyruvate (Burt et al., 1979). Certainly our intact brain sample does not show as strong a resonance as seen in in vivo human, rabbit and dog brains collected at field strength below 2 Tesla. Thus, if such compounds are present in the calf they are labile and break down within the 0.5-1 hr that the sample transportation requires (Delpy et al., 1982). The situation has become even more complex with the recent observation that the intensity of the peak in the phosphodiester region varies in in vivo preparation according to field strength (Sauter and Ruden, 1987). Higher field strengths do not show nearly as large an amplitude as that at lower fields. Since our measurements were made at a field strength comparable to the high field of Sauter and Rudan, an alternative explanation for the lower total intensity in the phosphodiester region could be due just to field strength. At any rate it is apparent that the nature of the total observed in vivo intensity in the phosphodiester region is a difficult and evolving question; however, we show here that the use of intact separated samples allows a simplification that makes analysis of such questions easier. Examination of the spectra show the presence of resonances in the monoester region. Comparison of published chemical shifts and extract work show that when two peaks are present they are phosphorylcholine (PCh) and phosphorylethanolamine (PEth). Our results confirm a published paper by Gyulai et al. (1984), that PEth is present in newborn dog brain. It also confirms previous classical biochemical results. The presence of more phosphomonoesters in gray matter than white could provide extra acidic buffering capacity that would go along with the suggestion for the observation that gray matter reaches a less acidic final pH than white (see Table 1). DISCUSSION TO our knowledge, this represents the first attempt to characterize by 31p NMR, gray and white matter. The results have appeared earlier in abstract form (Burt et al., 1986), and an even more recent abstract confirms in elderly humans the results given here: namely, phosphodiesters are higher in white matter than gray and vice versa for phosphomonoesters (Smith et al., 1988). Subsequent reports by others have featured proton N M R results of differences between gray and white matter (Petroff et al., 1987). In the latter report most of the metabolites observed seem to undergo modest temporal changes compared to phosphocreatine and ATP. This is in keeping with our observation that GPC is not substantially metabolized. Other factors which might effect the results have been enumerated. We have found therefore, that in calf brain, phosphodiesters are high in white matter. It has been noted in almost all neonates that PEth is high and then decreases with age. But in comparison to this study one cannot judge if this is due to a uniform decrease in PEth or to some kind of tissue differentiation. The method presented here should allow such questions to be addressed. We have not investigated whether our change persists with time, however, studies in two other species seem to indicate that it is
Phosphodiester distribution in the brain
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Table 2. Some phospholipid % concentration in differentiating systems State of PL PDE System differentiation % PL* content conc:~ Reference Muscle White Rabbit Extensor Digitorum Longus 90 Burt et al. (1982) Sarcoplasmic Reticulum Guinea Pig Vastus 64 13.5 Sarzala et al. (1982) Red Rabbit Soleus 70 + Butt et al. (1982) Sarcoplasmic Reticulum Guinea Pig Soleus 43 9.5 + Sarzala et al. (1982) Brain Gray (human) 72§ 37 ? Spector (1956) White (human) 51§ 85 ? Spector (1956) Astrocytomas (human-all types) 6211 15 ? Yates et al. (1979) Glioblastoma 5011 14 ? Yates et al. (1979) *Percent phospholipid expressed as (mg of phospholipid/g tissue divided by mg of total lipid per gram tissue) x 100. tWhere known phospholipid content expressed as mg/g tissue. :[:Phosphodiesterconcentration (PDE) graded on a relative scale from absent ( - ) to present ( + ). ? mark means data is not present. §Values represent midrange values from Spector's (1956) handbook. 11Basedon the % P and on an average mol wt calculated for a fixed fatty acid composition and the percent distribution of headgroups listed in Yates et al. (1979). a uniform phenomenon that persists into adulthood. Pettegrew has shown in whole rat brain extract that the phosphodiester to total phosphate ratio increases past birth and then remains high (Pettegrew e t al., 1986a). His results in terms of adult rats agree with those of Glonek e t al. (1982). In human surface coil studies it also appears that the phosphodiester region seems more prominent in adults than children (Bottomley e t al., 1984; Z i m m e r m a n et al., 1985). The human studies, of course, have the problem of field strength dependent phenomena mentioned previously. Nevertheless, for both cases an increase in phosphodiesters due to white matter proliferation is consistent with the data. Besides the N M R results from brain, it is also known that several other organs have phosphodiesters distributed in a very asymmetric manner. Thus G P C is high in kidney medulla but low in the cortex (Schimassek e t al., 1959). Phosphodiesters are more prominent in red muscle than white (Burt et al., 1982), and in surface coil studies are seen to increase disproportionately in Duchenne Dystrophy (Youkin et al., 1987). Table 2 tabulates these differences and also lists some of the lipid differences between differentiated tissues. In addition, our finding has direct relevance for studies using P C A extracts of brains. Any conclusion drawn about variations in phosphodiester concentration as characteristic of a condition or disease must bear in mind that different cerebral tissue may vary both with respect to percentage of gray and white matter or differentiation. In general, the larger the specimen being examined, the larger the artifacts could be, since it will contain a larger mixture of gray and white matter. H o w this can be applied to a specific problem is seen in studies on extracts of brains subject to dementia (Barany e t al., 1985; Miatto e t al., 1986). They found elevated G P C levels in pathological extracts, however, they do not account for possible variations in gray and white matter ratios. Since some studies show there is more atrophy of gray matter than white in aging, the expected relative increase in white matter together with the present study could
explain the elevated G P C levels on anatomical rather than physiological grounds, particularly if controls are not good age matches for the dementia cases (Duara et al., 1986). In terms of the biochemical significance of the appearance of GPC, it must be related to phospholipid metabolism. It has been suggested that phosphodiesters are mainly an index of catabolism (Pettegrew e t al., 1986b). In this regard one notes that total phospholipid concentration increases dramatically in white matter. F o r example, in the human, phospholipid concentration per gram of tissue is 85 m g / g m in white matter vs 37 in gray (Spector, 1956). So one could argue higher G P C levels could simply be due to more substrate. However, this is probably too simple a view for as pointed out by Ribolow and Burt (1987), G P C can serve as a lysophospholipase inhibitor. This means PP COl=Ch__
Fig. 4. Redrawn general scheme showing how the processing of phospholipids can be seen as a cycle. PC and LPC are phosphatidylcholine and lysophosphatidylcholine and are membrane-bound. The other -glycerol phosphoryleholine (GPC), ~-glycerol phosphate (ctGP), choline (Ch), phosphocholine (PCh), cytidine triphosphate (CTP), pyrophosphate (PP) and cytidine diphosphocholine (CDPCh) are all water soluble. The enzymes involved are: (I) phospholipase; (2) lysolecithin acyl transferase; (3) lysoelcithinase; (4) phosphodiesterase; (5) choline kinase; (6) phosphocholine-CMP transferase; (7) CDP choline transferase; and (8) phospholipase C. The reported inhibition of reaction 3 by GPC is shown by a dotted line from GPC. Other conceivable inhibitions by PCh (or PEth) are also marked.
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G P C may be helping to maintain high phospholipid levels by decreasing the rate of flow of phospholipid through lysophospholipid hydrolysis. Indeed from Table 2 it can be seen that regardless of total phospholipid concentration, phosphodiesters such as G P C are high in the differentiated tissue which has the lower % phospholipid composition. The scheme suggested above can be viewed in terms of Fig. 4 where the lipid soluble metabolites are seen to be connected in a cycle to the water soluble ones. It is suggested that control of the water-soluble G P C both by generation through lipases and breakdown through diesterases can control lipid-related events via feedback inhibition. Ribolow and Burt (1987) demonstrate this inhibition in sperm and seminal plasma with G P C at the 1 m M level. To summarize then, our research points out there can be substantial differences in the phosphorus spectra of gray and white matter at a particular stage of development in calf brain. It further adds brain to the list of tissues in which phosphodiesters serve as potential markers and modulators of development to brain. REFERENCES
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