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Capillary oxygen saturation and tissue oxygen pressure in the rat cortex at different stages of hypoxic

The objective of this study was to generate data that allow for estimation of the validity of oxygen saturation (S02)values in superficial cortical capillaries as calculated by a microreflectometric system (EMPHO /19). Capillary S02and tissue oxygen pressure 002) were measured simultaneously in the cortex of n = 13 Wistar rats undernormocapnic (PaCO2= 36 mmHg) arterial normoxia (PaO2= 92 mmHg), moderate (pa 02 = 53 mmHg) and severehypoxic hypoxia (PaO2 = 31 mmHg) with microreflectometry and multiwire surface electrodes. Values were pooledaccording to arterial oxygenation levels, displayed as frequency histograms and compared via ANOVA (pKeywords: Capillary oxygen saturation; tissue oxygen pressure; rat cortex; hypoxia; reflection-spectrophotometry;multiwire-surface electrodesINTRODUCTIONMonitoring of tissue oxygen supply and metabolism has gained widespread interest in the neuroscience community,because methods which have been applied in animal experiments for a very long time were made amenable toclinical research by technological advancements within the last years. There are two basic principles for thosemethods in clinical application.
Methods based on polarographic principles' on one hand measure tissue oxygen partial pressure (Pt02) and are nowmainly in use in the form of oxygen probes that are inserted in the brain tissue close to regions of interest. They areestablished monitoring devices for regional Pt02 in intensive care patients and during operative interventions2-4.
Recently developed sensors incorporate combinations of electrochemical and fiberoptic elements allowing forsimultaneous recording of regional tissue PO^sup 2^, PCO^sup 2^, pH and temperature.5-7 Other polarographictechniques such as multiwiresurface electrodes8-13, have never reached such acceptance in clinical research due totheir complex set-up, calibration procedure etc, which renders intro-operative use cumbersome and time-consuming14,15. Yet they are very useful for laboratory work, because they measure local organ surface PO^sub 2^ with avery high spatial resolution. Representative PtO^sub 2^-distributions can be obtained reflecting adequately theheterogeneity of oxygenation in the superficial cortical layers of the brain.
Optical sensor techniques on the other hand have also been used for studies of tissue oxygen metabolism for a verylong time providing information about oxygenation of hemoglobin or the redox state of intracellular enzymes' .
Many methods have been developed using different algorithms and wavelengths (e.g. infrared vs. visible light),which also differ basically with respect to their catchment volume (e.g. macroscopic vs. microscopic reflectancespectroscopy)21-24. Spatially resolved near-infrared spectroscopy systems (NIRS) are well established methodsnowadays, and are used for non-invasive transcranial monitoring of regional S02 (TCCO) in the clinical setting25-31. Microreflectometric systems work mainly on the domain of visible light. They are less frequently used clinicallythan in laboratory experiments 32. However, they can provide clinically important information about local S02-distributions in superficial cortical capillaries. We and others have used a high resolution microreflectometric system(EMPHO Il, BGT, Uberlingen, Germany) for in vivo measurements of capillary S02 in the human cortex . It hasbeen proven that the obtained S02 values reflect changes of nutritive capillary flow in the cortex of rats veryaccurately and with high sensitivity under constant conditions of arterial oxygen supply and consumption36. Themajor criticism of this technique lies in the fact that no exact validation procedure for brain tissue exists 36,37,although in vitro experiments have shown an excellent correlation with tissue oxygenation levels38.
The aim of this study was to generate data, which allow for a better estimation of the validity of SO^sub 2^ valuesobtained by this method in the brain cortex under various arterial oxygenation levels in vivo.
MATERIALS AND METHODSMeasurements of capillary cortical oxygen saturation (SO^sub 2^)Capillary SO^sub 2^ values were measured with the Erlangen Microlightguide Spectrophotometer (EMPHO)(R) II,BGT, Germany), which was introduced and described in 198939. It was designed for fast, diffuse remissionspectrophotometry by flexible microl ightgu ides in small tissue volumes of moving organs in situ. Light in thevisible domain illuminates tissue via the iluminating fiber and backscattered light is transmitted via six detectingfibers (0 70 (mu)um) arranged in a hexagonal pattern around the illuminating fiber, to a rotating band passinterference filter disc. This serves as a monochromating unit in the spectral range of 502-628 nm in 2 nm steps.
Spectra of 64 wavelengths per rotation are thus transmitted to a photo multiplier, an AD-converter and finally to acomputer, in which one SO^sub 2^ value per spectrum is calculated by an algorithm described elsewhere 40,41 .
The high temporal (100 spectra sec^sup -1^) and spatial (75*250 mum) resolution permits an easy scanningprocedure of superficial cortical capillaries by moving the light-guide above the brain surface.
Measurements of cortical tissue oxygen pressure (Pt02) Cortical Pt02 was measured with polarographic multiwiresurface electrodes described by Kessler and Lubbers 30 years ago42,43 (MIT-system Dortmund, Germany)containing eight platinum wires allowing eight simultaneous Pt02 measurements in superficial cortical brain tissuebased on the Clark principle'. The electrode is counterbalanced on a lightweight arm, permitting it to follow brainpulsations without exerting pressure. It is further mounted on a micromanipulator to allow for scanning proceduresby precise and free movement across the brain surface. The absolute Pt02 values are recorded on a first-generation PC system calculating frequency histograms and mean values. For a more detailed description of the complexcalibration and measurement principles of this well known technique we refer to the existing literature.
Experimental protocolThirteen male Wistar rats weighing between 350 and 400 g were anesthetized by 0.015 mg kg-' Fentanyl and 0.8 mgkg-' Droperidole i.m., maintained by 25% of the initial dose every 30 min throughout the experiments. The animalswere intubated via tracheostomy. Ventilation was controlled by using a small animal respirator to maintainnormocapnia (PaC02: .:;36 mmHg) during three different stages of arterial oxygenation obtained by changing theinspired 02 fraction (21%-- 10%- 4%) until a steady state was reached. The right femoral artery was cannulated forcontinuous blood pressure monitoring and intermittent blood gas analysis. Rectal temperature was controlled andmaintained by a heating pad and lamp at 380C.
The animals were mounted on a stereotactic head frame and a parietal craniotomy was carried out under themicroscope using a diamond drill. Thereafter the dura mater was stripped off and the brain surface was continuouslyrinsed with warm saline solution. After reaching a steady state for the corresponding Pa02, cortical capillary S02 andPt02 was measured by scanning the exposed parietal cortex in each animal under (a) normoxia (Pa02: ,92 mmHg),(b) moderate hypoxia (PaO2: 53 mmHg) and severe hypoxia (Pa02: .31 mmHg).
Data were transferred to a commercially available computer software for graphical and statistical analysis (ANOVA,pRESULTSPhysiological variables such as PaC02, MABP and rectal temperature were comparable during different stages ofarterial hyoxia respectively normoxia (Table 1). A total number of n=9617 S02 respectively n=1118 Pt02 values wasobtained during normoxia (Pa02=92.3 +2.2 mmHg) in all animals, n=7512 S02 respectively n=976 Pt02 valuesduring moderate hypoxia (PaO2=53.51.2 mmHg) and n=6175 S02 respectively n=788 PtO2 values during severehypoxia (Pa02=30.71.6 mmHg). The calculated mean values SD for pooled data of capillary oxygen saturation andtissue oxygen pressure in the parietal cortex were 45.6% + 14.6% SOZ respectively 26.8 + 8.2 mmHg PtO2 duringarterial normoxia, 32.6%10.2% SOZ respectively 20.26.6 mmHg PtO2 during moderate arterial hypoxia and12.3%11.1% S02 respectively 8.75.0 mmHg during severe arterial hypoxia (Table 2). Comparison of S02 and Pt02frequency histograms correspondingly showed a parallel shift to the left with decreasing arterial oxygenation (Figure1). During severe hypoxic hypoxia more than 40% of Pt02 values were below 10 mmHg and more than 80% of S02values below 25% SO2. Distributions of oxygen saturation and oxygen pressure values became more homogeneousfrom normoxia to moderate hypoxia indicating a reactive increase in regional cortical blood flow. In a Hill plot an'in vivo tissue oxygen dissociation curve' was accomplished with SOZ values ranging from 1 % to 65% SOZ andPtO2 values from 0.1 to 41 mmHg. Linear regression analysis showed an excellent correlation with a coefficient ofdetermination of 12 of 0.88 (Figure 2). However, on closer examination of the plot it became obvious that the scatterof values around the line of regression is definitely increased below 10% SOz respectively 1.5 mmHg Pt02.
DISCUSSIONThe microspectrophotometric system described in this article (EMPHO 10) will obviously never be applied as oftenas non-invasive NIRS-systems in clinical routine, because it requires surgical exposure of the cerebral cortex. Froma scientific point of view, however, both methods should be regarded as complementary, because they differbasically in their catchment volumes. Opposed to NIRS-systems the EMPHO II is able to provide information ontissue oxygenation in superficial cortical areas alone with very high spatial resolution. Its excellent temporalresolution allows for easy and fast scanning procedures rendering it thus a feasible tool during neurosurgicalintervention S33-35,37. Data acquisition is possible over any desired extent of the exposed cortex under direct visualcontrol of normal anatomy and accompanying pathologies. The obtained S02 distributions therefore accuratelyreflect variations in and/or natural heterogeneity of tissue oxygenation in relation to underlying cortical structures.
With these properties the system can offer new and valuable insights into the cortical microcirculation in animalexperiments as well as in clinical researchHowever, a potential source of error exists and has been acknowledged previously. The lack of an exact validationfor brain tissue might violate assumptions in the algorithm, which would be of interest for assessment of trulyabsolute cortical S02 values, although comparison of EMPHO III data with S02 values of other systems forintravital microreflectometry under comparable conditions in the rat cortex 32 suggests that this error is probablyminor. Therefore experiments have been performed to compare these S02 data with methods regarded as the goldstandard for tissue oxygenation, that is multiwire surface electrodes, which measure tissue oxygen partial pressure002) with approximately the same spatial resolution as microreflectometric systems (e.g. EMPHO 110)38. In thisexperimental setup jejunal mucosal oxygenation was measured with both methods simultaneously, and it wasdemonstrated that S02 and PtO2 values varied within the expected range, following in parallel major oscillationsinduced by vasomotion. To test the validity of the EMPHO II in an additional in vitro experiment S02 and Pt02values were obtained in a tissue homogenate under varying levels of arterial oxygenation. This produced a 'tissueoxygen dissociation curve', which showed an excellent correlation (r=0.95) of both values. However, values rangedbetween 20% and 80% S02 only, omitting the critical range below 20%. We thus reproduced the experiments in therat cortex in vivo within a lower range of arterial oxygenation levels to create a more realistic environment forcomparison of both methods under pathophysiological conditions. Our results regarding the Pt02 distributions in therat cortex under arterial normoxia and different stages of hypoxia are in full accordance with those of prior animal experiments using the same algorithm and methodology9-12,44. This holds true for mean values, shift ofdistributions and reaction to hypoxic-induced increase of local CBF 45,4s,as. The data derived from the multiwiresurface electrode measurements can therefore be regarded as reliable reference values under these experimentalconditions.
The stepwise decrease of arterial oxygenation produced an average desaturation from 46% to 33% to 12% S02within the cortical capillaries, which paralleled a decrease of oxygen partial pressure in the same cortical areas from27 to 20 to 9 mmHg Pt02. Thus a 30% drop of oxygen saturation in the vascular supply unit caused a 26% decreaseof oxygen tension within the depending cortex, respectively a further reduction of S02 of 60% a subsequent decreaseof Pt02 of 55%. The values calculated by the algorithm of the EMPHO II lie within the expected range of S02values, when compared with those for calculated 02 dissociation curves in brain capillaries under the given arterial02 partial pressures". Furthermore they correspond exactly to those described by Watanabe et al.32 in the brain ofhypoxic Wistar rats measured by an analogous microspectrophotometric system. However, the seemingly linearrelationship between S02 and Pt02 values in our experiment does not reflect the complicated interdependencies inreality and is most probably caused by the necessary averaging procedure for numerous values in multiplecapillaries and cortical areas supplied by them. In reality the relation between 02 saturation and 02 partial pressure inbrain capillaries is known to be sigmoid and the one between intracapillary 02 partial pressure and cortical PtO2exponential in models regarding one isolated capillary segment47-49. To approximate these complicated relations ina descriptive linear manner usually a Hill-plot is used as done by Hasibeder et a1.38 in their in vitro experiment. Wehave adopted this approach and have thus produced an 'in vivo tissue oxygen dissociation curve', which indeeddemonstrated a significant linear dependency with an excellent correlation. The coefficient of determination r2 wascalcualted as 0.88, which indicates that Pt02 values per se contain approximately 90% of all information needed topredict intracapillary SO, and vice versa.
Yet, apart from pure statistical calculations, it is obvious that the scatter of values around the line of regression is nothomogeneous (Figure 2). A definitive increase can be observed at its lower end, that is at values below 10% SOz,respectively 1.5 mmHg PtO2. The reason for this divergence at very low levels of oxygenation cannot be pinpointedexactly. The easiest explanation would be that the algorithm for calculation of S02 data is not valid at very lowoxygen levels despite the fact that the EMPHO II works on the basis of multiple wavelengths. This would be trueunder the assumption that PtO2 values by multiwire surface electrodes are the gold standard for reference. It is,however, a known fact that multiwire surface electrodes are neither sensitive nor stable enough to measure very lowPtO2 values with absolute precision,50. Exact quantification of values in this range remains somewhat arbitrarysince even the best of electrodes will show drift artefacts. Furthermore it is not clear whether the logarithmictransformation of the Hill plot is still valid to approximate the relation between S02 and PtO2 in a severely hypoxicsituation, because intracellular and intravascular changes in pH, PaCO2 and lactate levels etc. will influence 02capacity, 02 affinity, 02 diffusion and local CBF in an uncontrolled way", z,sz.
We could, therefore, show that S02 values in superficial cortical capillaries as calculated by the algorithm of theEMPHO III are highly accurate over a wide range of oxygenation levels. Only with respect to extremely low valuesdoes it remain questionable whether they reflect truly absolutes of capillary 02 saturation and one should thereforebe cautious in interpretation of single values in the range below 10% SO2. This shortcoming, however, is not uniqueto the method described here, but probably inherent to every method applied for data sampling in cerebrovascularmicrovolumes, including laser Doppler flowmetry. In microreflectometric measurements single values in general,irrespective of 02 saturation level, should never be regarded as representative. We and others have shownpreviously36,37, that to reach reliable and reproducible results scanning procedures are mandatory with thesemethods. Thus we consider only accumulations of low S02 values, that significantly exceed those found due tonatural heterogeneity of local CBF unequivocal indicators for cortical hypoxia or ischemia.
We conclude that the EMPHO II is a highly reliable and feasible instrument for experimental and clinicalcerebrovascular research, if information about cortical capillary 02 saturation with high spatial and temporalresolution is desired.
REFERENCES1 Clark L, Wolf R, Granger D, Taylor Z. Continuous recording of blood oxygen tensions by polarography. J ApplPhysiol 1953; 6: 189-1932 van Santbrink H, Maas Al, Avezaat CJ. Continuous monitoring of partial pressure of brain tissue oxygen inpatients with severe head injury. Neurosurgery 1996; 38: 21-313 Meixensberger J, Baunach S, Amschler J, Dings J, Roosen K. Influence of body position on tissue-p02, cerebralperfusion pressure and intracranial pressure in patients with acute brain injury. Neurol Res 1997; 19: 249-2534 Dings J, Meixensberger J, Jager A, Roosen K. Clinical experience with 118 brain tissue oxygen partial pressurecatheter probes. Neurosurgery 1998; 43: 1082-10955 Hoffman WE, Charbel FT, Edelman G, Abood C. Brain tissue response to COZ in patients with arteriovenousmalformation. J Cereb Blood Flow Metab 1996; 16: 1383-13866 Hoffman WE, Charbel FT, Edelman G, Ausman JI. Thiopental and desflurane treatment for brain protection.
Neurosurgery 1998; 43: 1050-1053 7 Hoffman WE, Charbel FT, Portillo GG, Edelman G, Ausman JI. Regional tissue P02, PCO^sub 2^, pH andtemperature measurement. Neurol Res 1998; 20: S81-S848 Crockard HA, Symon L, Branston NM, Juhasz J, Wahid A. Measurements of oxygen tension in the cerebral cortexof baboons. J Neurol Sci 1976; 27: 17-289 Grote J, Zimmer K, Schubert R. Effects of severe arterial hypocapnia on regional blood flow regulation, tissuePOz and metabolism in the brain cortex of cats. Pflugers Arch 1981; 391: 195-19910 Grote J, Zimmer K, Schubert R. Tissue oxygenation in normal and edematous brain cortex during arterialhypocapnia. Adv Exp Med Biol 1984; 180: 179-18411 Leniger-Follert E, Lubbers DW, Wrabetz W. Regulation of local tissue POz of the brain cortex at differentarterial 02 pressure. Pflugers Arch 1975; 359: 81-9512 Leniger-Follert E, Lubbers DW. Behavior of microflow and local P02 of the brain cortex during and after directelectrical stimulation. A contribution to the problem of metabolic regulation of microcirculation in the brain.
Pflugers Arch 1976; 366: 39-4413 Leniger-Follert E. Direct determination of local oxygen consumption of the brain cortex in vivo. Pflugers Arch1977; 372: 175-17914 Assad F, Schultheibeta R, Leniger-Follert E, WOllenweber R. Measurements of local oxygen partial pressure(P02) of the brain cortex in cases of brain tumors. Adv Neurosurg 1984; 12: 263-27015 Schultheiss R, Leuwer R, Leniger-Follert E, Wassmann H, Wullenwebert R. Tissue P02 of human brain cortex-method, basic results and effects of pentoxifylline. Angiology 1987; 38: 221-22516 Chance B. Spectrophotometry of intracellular respiratory pigments. Science 1954; 120: 767-77417 Jobsis FF. Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatoryparameters. Science 1977; 198:1264-126718 Jobsis FF, Keizer JH, LaManna JC, Rosenthal M. Reflectance spectrophotometry of cytochrome aa3 in vivo. JAppl Physiol 1977; 43:858-87219 Kariman K, Burkhart DS. Non-invasive in vivo spectrophotometric monitoring of brain cytochrome aa3revisited. Brain Res 1985; 360: 203-21320 Bashford CL, Barlow CH, Chance B, Haselgrove J, Sorge J. Optical measurements of oxygen delivery andconsumption in gerbil cerebral cortex. Am J Physiol 1982; 242: C265-C27121 Kinuta Y, Kikuchi H, Ishikawa M, Hirai 0, Imataka K, Kobayashi S. Reflectance spectrophotometricmeasurement of in vivo local oxygen consumption in the cerebral cortex. J Cereb Blood Flow Metab 1987; 7: 592-59822 Narita N, Tominaga T, Koshu K, Miozoi K, Yoshimoto T. Monitoring of brain tissue hemoglobin concentrationand oxygen saturation using a three wavelength spectrophotometric method. Neurol Res 1994; 16: 428-43223 Newton DJ, Harrison DK, Delaney CJ, Beck JS, McCollum PT. Comparison of macro- and micro-lightguidespectrophotometric measurements of microvascular haemoglobin oxygenation in the tuberculin reaction in normalhuman skin. Physiol Meas 1994; 15: 115-12824 Tateishi N, Maeda N, Shiga T. A method for measuring the rate of oxygen release from single microvessels. CircRes 1992; 70: 812-81925 Ausman JI, McCormick PW, Stewart M, et al. Cerebral oxygen metabolism during hypothermic circulatory arrestin humans. J Neurosurg 1993; 79: 810-81526 Dujovny M, Ausman JI, Stoddart H, Slavin KV, Lewis GD, Widman R. Somanetics INVOS 3100 cerebraloximeter [letter, comment]. Neurosurgery 1995; 37: 16027 Hock C, Muller-Spahn F, Schuh-Hofer S, Hofmann M, Dirnagl U, Villringer A. Age dependency of changes incerebral hemoglobin oxygenation during brain activation: A near-infrared spectrosocpy study. J Cereb Blood FlowMetab 1995; 15: 1103-110828 Kirkpatrick PJ, Smielewski P, Czosnyka M, Menon DK, Pickard JD. Near-infrared spectroscopy use in patientswith head injury. I Neurosurg 1995; 83: 963-97029 McCormick PW, Stewart M, Goetting MG, Balakrishnan G. Regional cerebrovascular oxygen saturationmeasured by optical spectroscopy in humans. Stroke 1991; 22: 596-60230 Samra SK, Dorje P, Zelenock GB, Stanley JC. Cerebral oximetry in patients undergoing carotid endarterectomyunder regional anesthesia. Stroke 1996; 27: 49-5531 Smielewski P, Kirkpatrick P, Minhas P, Pickard JD, Czosnyka M. Can cerebrovascular reactivity be measuredwith near-infrared spectroscopy? Stroke 1995; 26: 2285-229232 Watanabe M, Harada N, Kosaka H, Shiga T. Intravital microreflectometry of individual pial vessels and capillaryregion of rat. J Cereb Blood Flow Metab 1994; 14: 75-8433 Hoper J, Gaab MR, Batz M, Feyerherd F. Local oxygen supply to the cerebral cortex during thiopental andpropofol anesthesia. First results. Anaesthetist 1994; 43: 534-53834 Hoper J, Gaab MR. Effect of arterial PCO2 on local HbO2 and relative Hb concentration in the human brain - astudy with the Erlangen micro-lightguide spectrophotometer (EMPHO). Physiol Meas 1994; 15: 107-11335 Meyer B, Schaller C, Frenkel C, Schramm J. Physiological steal around AVMs of the brain is not equivalent tocortical ischemia. Neurol Res 1998; 20: SI 3-S1 7 36 Nakase H, Heimann A, Kempski 0. Alterations of regional cerebral blood flow and oxygen saturation in a ratsinus-vein thrombosis model. Stroke 1996; 27: 720-72737 Meyer 13, Schaller C, Frenkel C, Ebeling B, Schramm J. Distributions of local oxygen saturation and its responseto changes of mean arterial blood pressure in the cerebral cortex adjacent to arteriovenous malformations. Stroke1999; 30: 2623-263038 Hasibeder W, Germann R, Sparr H, et al. Vasomotion induces regular major oscillations in jejunal mucosal tissueoxygenation. Am J Physiol 1994; 266: 6978-698639 Frank KH, Kessler M, Appelbaum K, Dummler W. The Erlangen micro-lightguide spectrophotometer EMPHO I.
Phys Med Biol 1989; 34: 1883-190040 Kubelka P, Munk F. Ein Beitrag zur Optik der Farbanstriche. Z Technische Physik 1931; 11: 76-7741 Frank KH, Kessler M, Appelbaum K, Albrecht HP, Mauch ED. Measurements of angular distributions ofRayleigh and Mie scattering events in biological models. Phys Med Biol 1989; 34: 1901-191642 Kessler M, Grunwald W. Possibilities of measuring oxygen pressure fields in tissue by multiwire surfaceelectrodes. Progr Resp Res 1969; 3: 147-15243 Lubbers D, Baumgartl H, Fabel H, et al. Principle and construction of various platinum electrodes. Progr RespRes 1969; 3: 136-146 44 Grote J, Reulen JJ, Schubert R. Increased tissue water in the brain:Influence on regional cerebral blood flow and oxygen supply. Pathology of cerebrospinal microcirculation. AdvNeurol 1978; 20: 333-33945 Bewrecski D, Wei L, Otsuka T, et al. Hypoxia increases velocity of blood flow through parenchymalmicrovascular systems in rat brain. J Cereb Blood Flow Metab 1993; 13: 475-48646 Kozniewska E, Weller L, Hoper J, Harrison DK, Kessler M. Cerebrocortical microcirculation in different stagesof hypoxic hypoxia. J Cereb Blood Flow Metab 1987; 7: 464-47047 Astrup P, Engel K, Severinghaus J, Munson E. The influence of temperature and pH on the dissociation curve ofoxyhemoglobin in human blood. Scand Clin Lab Invest 1965; 17: 515-52348 Krogh A. The rate of diffusion of gases through animal tissue with some remarks on the coefficient of invasion. JPhysiol (Lond) 1918/ 19; 52: 39149 Grote J, Siegel G, Zimmer K, Adler A. The interaction between oxygen and vascular wall. Adv Exp Med Biol1989; 248: 575-581 50 Lubbers DW, Baumgartl H, Zimelka W. Heterogeneity and stabilityof local POz distribution within the brain tissue. Adv Exp Med Biol 1994; 345:567-574Bernhard Meyer, Rolf Schultheibeta and Johannes SchrammDepartment of Neurosurgery, University of Bonn, GermanyCorrespondence and reprint requests to: Bernhard Meyer, MD, Department of Neurosurgery, University of Bonn,Sigmund Freud Str. 25, 53127 Bonn, Germany. Accepted for publication June 2000.
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