INFRARED SPECTRA OF THE BRAIN AND LIPIDS$^{\ast}$

Author Institution: Divisions of Biochemistry and Neuropathology, Philadelphia General HospitalPrevious studies demonstrated that brain tissue from various locations shows characteristic infrared spectra which are species unspecific. The present paper reports: 1. Qualitative and quantitative spectroscopy of frozen and dried sections of brain tissue from adult and fetal rats. 2. Tentative assignment of brain tissue spectra. 3. Spectroscopic studies of an effect of ionizing irradiation on the brain. A large number of sections of brain tissue showed two different types of vibrations within the different wavelength region. a. In the high frequency region, below $8.1 \mu$, sections from the adult animal, fetus, and newborn alike showed a number of more or less strong bands at almost identical locations. These bands were quite similar to known vibrations ($3.04 \mu N H$ stretching, $3.4 \mu C H$ bending, $6 \mu CO$ stretching, the deformation vibration of N H at $6.44 \mu$ and the deformation vibrations of C H at $6.88 \mu$ and $7.25 \mu$) found in proteins and polypeptides (Sutherland). An additional medium strong band, still unassigned, could be regularly seen at $8.04-8.08 \mu$. b. In the low frequency region, e.g., between 8.10 and $11 \mu$, the spectra of adult and fetal brain tissue showed significant differences. In adult rats rather strong bands at $9.30-9.35 \mu$ and weaker bands at about $10.30 \mu$ were found. In the fetus and newborn, weaker bands characteristically occurred at about $9.50 \mu, 9.23 \mu$, and $10.30-10.34 \mu$. Lipids are contained in neural tissue in so high concentration that it seemed reasonable to investigate their role in producing the characteristic spectra of brain tissue. Study of brain sections before and after extraction with various lipid solvents (mixtures of chloroform: ether (1:1), petroleum ether: methanol (9:1), or ethanol) demonstrated the correctness of this assumption. Ether: chloroform extraction greatly reduced the absorption in the $9-11 \mu$ region and shifted the strong band found in adult brain tissue at $9.30-9.35 \mu$ toward longer wavelength, thus giving a spectrum almost similar to that of fetal brain tissue. The protein polyamide vibrations such as the deformation vibration of N H at $6.44 \mu$ were not affected by this extraction. Extraction with ethanol had even greater effect on the absorption bands in the $9-11 \mu$ region, these bands nearly completely disappearing after this extraction. Studies of pure compounds such as cholesterol, cholesterol ester, sphingosine, sphingomyelin, cerebrosides, kerasine, phrenosine, lecithin, cephalins, and cerebronic acid demonstrated that these compounds give more or less strong absorption bands in the $9-11 \mu$ region. Further assignment studies are in progress. The finding that chloroform: ether extraction removed only a practically amide-free lipid fraction was further proved by examination of pooled brain extracts, this fact was then used as a basis for quantitative estimation of the relative amounts of this ``free lipid fraction.'' Spectra of brain sections were recorded before and after the extraction. Optical densities were determined in the bands at 10.30, 9.30-9.35, and about $6.44 \mu$ in adult brain and at 10.34, 9.50, and about $6.44 \mu$ in fetal and newborn brains. Ratios between densities of the characteristic bands in the $9-11 \mu$ region and the densities of the deformation vibration of N H at $6.44 \mu$, used as internal standard, were calculated. The differences of the ratios of densities before and after the extraction represent a relative estimate of the ``free lipid fraction.'' The determinations in the fetus and newborn gave significantly different results from those in adult rats indicating a considerable smaller amount of this lipid fraction in developing brain. Study of large numbers of brain sections with this technique show, moreover, that a definite pattern exists in the adult brain. The brain stem presents the highest, and the forebrain, the lowest differences of ratios of optical densities, with hypothalamus, midbrain, and cerebellum between the two extremes. This procedure was now applied to a study of the effect of ionizing irradiation upon the brain of fetal and adult rats. A large number of adult female rats were subjected to total body irradiation with doses varying from 500 to 800 r, pregnant animals, with doses from 150 to 400 r. The animals were killed 1 to 2 days after the irradiation. The differences of ratios of optical densities were determined in sections through brain stem, hypothalamus, midbrain, cerebellum, and forebrain of the adult animals and in sections through the whole brain of a number of litter mates obtained from each pregnant irradiated animal. The results, compared with appropriate controls, indicate that ionizing irradiation produces different effects on the brain tissue of adult and fetal animals. Irradiated adult rats, showed a small but significant lowering of the free lipid fraction, especially in cerebellum and forebrain; irradiated fetal rats, on the other hand, showed a definite increase of that lipid fraction when compared with nonirradiated controls. These results may contribute to the elucidation of the greater radiosensitivity of the fetal brain recently demonstrated (Hicks) and confirmed our own histologic studies. Summarizing the general results and implications of this investigation, it is felt that although much more work on assignment of lipid spectra and the various lipid fractions of the brain is needed, the present data already indicate the value of infrared spectroscopy for the study of some of the complicated problems involving different lipids in the brain