Analysis of micellar and vesicular lecithin and cholesterol in model bile using 1H- and 31P-NMR.

The distribution of phospholipid and cholesterol between the vesicular and micellar phases in bile plays an important role in the formation of cholesterol gallstones. Conventional analytical procedures to determine the distribution are potentially unreliable because they disturb the distribution of these compounds between the two phases. In this work, we circumvent this problem by using NMR. 31P-NMR is used to quantify directly the micellar and the vesicular amount of lecithin. The previously described 1H-NMR method to determine directly the micellar lecithin (Groen et al., J Lipid Res 31: 1315-1321) has been optimized by the implementation of a spectral quantification method. The agreement between the 31P and 1H methods was excellent. In our hands, the method of Ellul et al. (FEBS Lett 300: 30-32) did not allow quantification of micellar cholesterol, although our fitting procedure offered the possibility of quantifying overlapping spectral peaks by the use of prior knowledge about all the parameters of the compounds visible in the spectrum. The micellar cholesterol concentration was so low compared to the overlapping lecithin peaks that no reliable quantification was possible. The same problem was encountered when using other characteristic cholesterol resonances for quantification. Our data suggest that cholesterol present in the vesicular phases is too immobile to give rise to high-resolution peaks and the amount of cholesterol present in the micellar phase is too low to allow quantitation by NMR. We conclude that 1H-NMR can be used to determine micellar lecithin, and 31P-NMR to determine micellar as well as vesicular lecithin in model bile.

In vivo 31P NMR spectroscopy of the rat cerebral cortex during acute hepatic encephalopathy.

During the development of acute hepatic encephalopathy, induced by acute liver ischemia, changes in brain 31P NMR spectra and EEG spectra were studied over 8:45 h in eight rats. At the end of this period the brain amino acid concentrations were determined. The results were compared with the same measurements in four normal and three portacaval shunted rats. Signs of acute HE, as judged by the EEG left index, started 5 h after the induction of acute liver ischemia. No accompanying significant changes in the cortical relative phosphocreatine and ATP concentration and intracellular pH were observed. The cortical relative Pi concentration had only slightly increased at t = 8 h. The concentrations of almost all measured brain amino acids, especially glutamine had increased at t = 8:45 h. At t = 8 h, rats with very severe HE had a small, but significant decrease of brain ATP concentrations. Their brain amino acid concentrations were more disturbed than in rats with less severe HE. It is concluded that a change in the cortical cerebral energy rich phosphate concentration is not an important pathophysiological mechanism during the development of acute HE. The observed changes in brain amino acids concentrations could be either part of a multifactorial pathogenesis or could be epiphenomena.

Murine mammary tumor response to hyperthermia and radiotherapy evaluated by in vivo 31P-nuclear magnetic resonance spectroscopy.

The response of the s.c.-implanted murine mammary carcinoma NU-82 to hyperthermia was followed as a function of time by 31P-nuclear magnetic resonance spectroscopy. Treatment consisted of elevation of the temperature of the tumors to 41-45 degrees C during 15 min. At 18 h after temperatures of up to 42, 43, 44, and 45 degrees C the ratio of ATP/Pi was unchanged, decreased, largely decreased, and approaching zero, respectively. After the higher doses the relative concentrations (in percentage of total phosphate as visible in the nuclear magnetic resonance spectrum) of phosphomonoesters (mainly phosphoethanolamine) and phosphocreatine also decreased in favor of Pi. The changes in phosphodiesters (mainly glycerophosphocholine) correlated linearly with the changes in ATP (r = 0.84, P less than 0.025). Whereas the limited spectral changes after a dose of 43 degrees C were nullified within 24 h, the more drastic changes after a dose of 45 degrees C lasted at least 8 days. The heavier dose not only induced temporary decreases in tumor perfusion like the lower dose (phase 1) but subsequently, unlike the lower dose, resulted in formation of necrosis (phase 2). In the same tumor we found increases in Pi and decreases in ATP and phosphodiesters after radiotherapy with a dose of 20 Gy. Radiotherapy (20 Gy) combined with hyperthermia (44 degrees C) appeared to strengthen these effects and resulted in an improved tumor response (regression).

Brain 31P NMR spectroscopy in the conscious rat.

Using a home-built head-body holder (HBH) which enables in vivo 31P NMR spectroscopy measurements on conscious rats, no significant changes were observed in the cerebral relative concentrations of ATP, phosphocreatine, phosphomonoesters, inorganic phosphate and intracellular pH during pentobarbital anesthesia in normal rats as compared to their conscious state.