Scyllo-inositol depletion in hepatic encephalopathy

Life Sciences, Vol. 54, No. 20, pp. 150%1512, 1994 Copyright © 1994 Elsevier Science lad Printed in the USA. All rights rtservr,d 0024-3205/94 $6.00 + .00

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SCYLLO-INOSITOL DEPLETION IN HEPATIC ENCEPHALOPATHY Yeong-Hau H. Lien +, Thomas Michaelis*, Rex A. Moats*, and Brian D. Ross* +Department of Medicine, University of Arizona Tucson, AZ 85724 • Huntington Medical Research Institutes Pasadena, CA 91105 and California Institute of Technology Pasadena, CA 91125 (Received in final form March 1, 1994)

Summary_ Cerebral myo-inositol depletion is found in patients with hepatic encephalopathy and can be implicated in the pathogenesis of hepatic encephalopathy. We measured scyllo-inositol, a stereoisomer of myo-inositol, in brain extracts from patients dying in hepatic coma using HPLC and high resolution ~H MRS. The cerebral scyllo-inositol concentration, determined by both methods, in patients without hepatic encephalopathy was 0.41+_0.11 mmol/kg wet weight. It decreased by 73% and 76%, respectively, as measured by HPLC and 'H MRS, in patients with hepatic encephalopathy. These findings indicate that myo-inositol depletion in patients with hepatic encephalopathy is not due to enhanced conversion of myo-inositol to scylloinositol or inhibition of myo-inositol transport by scyllo-inositol, but rather to the reduced biosynthesis or transport of both inositols. Key Words: scyllo-inositol,hepatic encephalopathy,myo-inositol

Hepatic encephalopathy (HE) is associated with many biochemical changes in the brain including an increase in glutamine concentration, decrease in glucose utilization, increase in blood-brain amino acid transport, and increase in synthesis and turnover of serotonin (14). Recently, using in vivo ~H magnetic resonance spectroscopy (MRS), Kreis et al (5,6) reported a new biochemical deficit, cerebral myo-inositol (MI) depletion in patients with HE. These changes were further confirmed in autopsied brain samples from patients dying in hepatic coma and in the brain of portacaval shunted rats using high performance liquid chromatography (HPLC) and in vitro IH MRS (7). Scyllo-inositol (SI), a stereoisomer of MI, is also present in the central nervous system (8). Gas chromatography analysis of normal human brain determined the MI/SI ratio of about 20:1 (9). The role of SI in the cerebral metabolism is unknown, and it is suggested that its presence in the brain is simply because of its similar affinity to the inositol transport system as MI (10,11). Recently, Strieleman et al (12) reported that addition of SI to cultured media significantly reduced MI transport and phosphoinositide concentrations in rat embryos, and caused growth and developmental retardation as well as malformations in both neural and extraneural tissues. In the case of HE, in addition to MI depletion, Kreis et al (5) demonstrated a significant reduction of an unassigned resonance (3.35 ppm) in the difference spectra obtained by subtracting Correspondence: Dr. Yeong-Hau H. Lien, Renal Section, Department of Medicine University of Arizona Health Sciences Center. Tucson AZ 85724

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normal spectrum from the spectra of HE patients. This resonance was recently assigned by Michaelis et al (13) as SI in 'H MR spectra of cerebral tissue in vitro and in vivo. The purpose of this in vitro study was to confirm the suggestive in v&o MRS findings that SI is depleted in HE, using two independent quantitative methods, and thereby, to exclude the possibility that MI depletion is due to enhanced conversion of MI to SI or inhibition of MI transport by elevated cerebral SI concentration. Methods

Twelve autopsied brain samples were obtained from two cirrhotic patients dying in hepatic coma and three control patients dying of pulmonary embolism, respiratory collapse, and myocardial infarction. Samples were dissected from the frontal, parietal or occipital lobes and frozen in liquid nitrogen and stored at -70°C. The time interval from death to freezing of dissected autopsied material was in a range of from 12 to 24 hours. Weighed cerebral tissue was homogenized in 6% perchloric acid (PCA), neutralized with 30% potassium hydroxide. PCA extracts for ~H MRS study were lyophilized and dissolved in D20 containing 5 mM 3(trimethyl-silyl)l-(propanesulfonic acid) (DSS) (Sigma, St Louis, MO, USA), an internal chemical shift and concentration reference. High resolution 'H MR spectra were obtained on a AMX-500 NMR spectrometer (Bruker, Karlsruhe, Germany). The acquisition consisted of 128 scans with a repetition time of 5.0 s. The 16 K data set was zerofilled to 32 K. After exponential line-broadening of 0.05 Hz, the data were Fourier's transformed. The cerebral SI concentrations were quantitated by comparing the resonance areas of SI (3.35 ppm) and of the internal reference with respect to the dilution factor of each sample and expressed as mmol/kg wet weight. For HPLC study, PCA extracts were passed through a Sep-Pak C18 cartridge (Millipore, Milford, MA, USA) (14). HPLC was performed using a Sugar-Pak 1 column (Waters Associates, Milford, MA) as described by Lien et al (15). An aliquot of 50/xl of brain extract was injected and eluted with 0.1 mM calcium disodium EDTA at 0.5 ml/min and 84°C. Metabolites were detected with a refractive index detector (Alter Scientific, Inc., Berkeley, CA, USA) and analyzed using an integrator. To verify the SI peak in brain extract, sample was spiked with SI (Sigma, St Louis, MO, USA). The concentration of SI in each brain sample was calculated from the peak area, adjusted for the dilution factors, and expressed as mmol/kg wet weight. Results

Fig. 1. shows 'H MR spectra of brain PCA extracts from a control patient (1A) and a HE patient (1B). Both 'H MR spectra were scaled to the internal reference and were corrected for their dilution factor. The reduction in SI and MI peak areas is obvious in the extract spectrum from HE brain. Although only a narrow frequency range is shown in the Fig. 1, several other resonances of metabolites could be detected. Glutamine was found markedly increased, and choline-containing compounds decreased in the brain extract from HE patient as reported previously (5,6). It is noteworthy, that taurine was not involved in the changes occurring with HE. Therefore, this metabolite is not responsible for the spectral changes (at 3.35 ppm) observed in 'H MR spectra of HE patients in vivo.

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Fig. 2 shows representative HPLC chromatograms of brain PCA extracts from a HE patient (2A) and a control patient (2B). When the control brain extract was spiked with 1/zg SI, only a peak with a retention time of 10.3 min was increased (2C). Thus, this peak was assigned as deriving from SI. The larger peak with a retention time of 11.8 min has been identified as MI previously (14). When compared with control HPLC chromatogram, both SI and MI peaks were significantly reduced in the brain extracts of HE patient. Fig 3. summarizes the results of cerebral SI concentration measurements by HPLC and in vitro 'H MRS in 12 samples from 3 control patients (2 brain sections each) and from 2 patients with HE (3 sections each). The normal cerebral SI concentration (0.41 ___0.11 mmol/kg wet weight by both HPLC and ~H MRS) was in excellent agreement with earlier quantification in vitro (9, 13) and in vivo (16). The cerebral SI concentrations in HE patients decreased dramatically (0.11 _ 0.08 mmol/kg wet weight by HPLC, and 0.10_+ 0.05 by q-I MRS, both p < 0.01 vs normal). The cerebral MI/SI ratio was 17:1 in the control group which was consistent with the previous report by Narumi et al (9). It was not possible to calculate this ratio in the HE group because there were two samples with unmeasurable SI levels by HPLC and one by MRS. When these samples were excluded, the MI/S! ratio was 24: I in the HE group which was not significantly different from that in the control group (p > 0.05).

Discussion We have demonstrated that cerebral concentration of SI is decreased significantly in patients with HE, when compared with that in patients without HE. Our in vitro study confirmed the previous suggestive in vivo tH MRS findings (5). Both SI and MI are accumulated in the brain by transport and synthesis. SI and MI share the same sodium-dependent inositol transport system in the brain with a similar affinity (10,11). MI can be synthesized from glucose by converting glucose-6-phosphate to inositol-l-phosphate which is mediated by inositol 1-phosphate synthetase [EC.5.5.1.4.] (17,18). SI can then be synthesized from MI by a NADP-dependent inositol isomerase in the brain (19). The finding that cerebral SI is depleted essentially excludes SI as a cause of MI depletion in HE patients. It is possible that the decrease in both MI and SI is due to reduced blood-brain inositol transport in HE patients. Alternatively, the decrease of MI and SI may be due to decreased synthesis in the brain, or in other tissues which are believed to perform this biosynthesis. The synthesis of MI and its conversion to SI may be suppressed in HE because of decreased glucose utilization as demonstrated in hyperammonemic animals by Mans et al (2). The pathological significance of SI depletion in HE is unclear. Depletion of SI content in nerve tissue has been reported in rats fed with a 40% galactose diet for 2 months (20). The nerve MI falls to one-forth and SI falls to one-tenth of the normal tissue levels. Following removal from galactose diet both MI and SI can be recovered in parallel. Michaelis et al (13) have reported that the cerebral SI resonance is increased significantly in a 'H NMR spectrum of a 15-months-old boy with Leigh's disease along with an increase in MI resonance. Parallel changes in MI and SI tissue contents are found in a number of pathologies, indicating their close metabolic relationship (13, personal communication, Ross, Moats, and Michaelis). Further investigation in this area may lead to a better understanding of the role of SI in the central nervous system. In summary, we used two independent methods for quantification of tissue SI concentration and for the first time demonstrated that SI, like MI, is depleted in patientswith HE. The poorly

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Fig. 1 Representative 'H MR spectra of brain extracts from a control patient (1A) and a patient with hepatic encephalopathy (1B). Resonances of DSS (*), creatine, choline-containing compounds (Choline), myo-inositol (mI), scylloinositol, taurine, and glutamine are indicated. The spectra are plotted normalized to the internal reference and corrected for extract dilution factors.

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lnositols in Hepatic Encephalopathy

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understood cerebral inositol pathway is of importance in neurotransmission, hormone responsiveness and osmotic control in the central nervous system. The significance and mechanisms of SI and MI depletion in HE remain to be determined.

Acknowledgments This research was supported by NIH grant 1RO1DK45666-01 and Southern Arizona Foundation Research Award (YHL) and by the L.K. Whitter Foundation (BDR). TM is a Bosewell Fellow of the California Institute of Technology.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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