Formol Titration and Fornix

FORMOL TITRATION [Sorensen method, quantitative determination of amino acids] is based on the fact that when an excess of a neutral formalin solution acts upon a solution of amino acids or peptides at a very weakly acidic reaction (pH = 6.8), the amino groups are bound; the reaction can apparently be expressed by the equation R CH NH₂

R.CH-N:CH₂

I

+H₂O.

The amino groups are converted into neutral methylene groups, and the remaining free carboxyl groups are titrated with alkali (phenolphthalein indicator). Necessary reagents: 1) n/5 sodium hydroxide solution; 2) n/5 hydrochloric acid solution; 3) phenolphthalein solution: 0.5 g of phenolphthalein is dissolved in a mixture of 50 cm³ of alcohol and 50 cm³ of water; 4) sensitive litmus paper; 5) formalin solution, freshly prepared: to 50 cm³ of commercial formalin (30-40%) add 1 cm³ of phenolphthalein solution and n/5 alkali solution until a very faint pink color is obtained. The determination is performed in parallel with a control solution in vessels identical in shape and size. During titration, three stages are distinguished: Stage I corresponds to pH=8.3; II-pH=8.8; III-pH=9.1. The stages are established by the control, with the color of which the color of the test solution is compared. Determination: to 20 cm³ of water boiled to remove CO₂ (control), add 10 cm³ of formalin solution (5) and 5 cm³ of n/5 alkali solution; then, titrating back with n/5 HCl solution, bring the color to a faint pink (Stage I). To 20 cm³ of the test liquid, add 10 cm³ of formalin solution and then, by alternately adding n/5 solutions of alkali and hydrochloric acid, bring its color to the color of the control. After this, add 1 drop of alkali to the control solution—a clearly pink coloration appears (Stage II), then add 2 drops of alkali (Stage III)—the liquid turns an intense pink color. In parallel, by adding alkali and acid, bring the test liquid first to the second stage, then to the third. The volume of the control liquid must be the same as the test liquid at the end of the titration. Calculation: from the number of cubic centimeters of n/5 alkali used for the titration of the test liquid to Stage II, subtract the number of cubic centimeters of added n/5 hydrochloric acid, as well as the excess alkali added to the control solution. Each cubic centimeter of the obtained difference corresponds to 2.8 mg of N, determined by formol titration. If the test liquid contains ammonia (at a concentration greater than 0.02 molar), carbonic acid, or phosphoric acid, they must be removed beforehand, as they interfere with the determination. Ammonia is removed by distillation from liquid alkalized with calcined magnesia or CaO in a vacuum at a temperature not exceeding 40°. From protein hydrolysate, NH₃ is usually not removed, as its content there is insignificant. To remove phosphates and carbonic acid, add solid BaCl₂ (2 g per 50 cm³ of solution), phenolphthalein, and a saturated solution of Ba(OH)₂ to the liquid until a pink color appears, and then add another 5 cm³. After bringing to a certain volume with water, filter after 15 minutes through a dry filter, bring the filtrate to a neutral reaction, and process as usual. When working with strongly colored liquids, decolorize them as follows: to 25 cm³ of solution in a 50 cm³ volumetric flask, add enough HCl or NaOH so that the acid concentration in the solution is n/10, add 4 cm³ of 2N BaCl₂, and then, drop by drop while shaking vigorously, 20 cm³ of n/3 AgNO₃ solution; after the foam that forms subsides, dilute to the mark with distilled water freed of carbonic acid by boiling, shake, and filter through a dry filter. Before titration, the solution is neutralized to litmus. If the test solution is only slightly colored, the control liquid can be tinted accordingly by adding tropaeolin 0 or 00, methyl violet, Bismarck brown, etc. Formol titration gives incorrect figures for the following amino acids: for tyrosine, the figures obtained are too high; for proline, too low; arginine salt is titrated as a monobasic acid, because guanidine salts (just like urea) remain neutral even after the addition of formalin.

M. Karyagina.

FORNIX, an association-commissural system located in the brain; it consists mainly of longitudinal fibers arranged in the form of two compact arcuate bundles connected to each other in the middle part (Fig. 1). This plate, or body

Figure 1. 1-corpus callosum, a-genu, b-rostrum; 2-septum pellucidum; 3-trigonum fornicis; 4-commissura ant.; 5-lamina suboptica; 6-corp. mamillare; 7-hypophysis; 7'-tuber cinereum; 8-chiasma; 9-infundibulum; 10-pedunculus cerebri; 10'-subst. nigra; 11-commiss. post.; 12-thalamus opticus; 12'-pulvinar; 13-commiss. grisea; 14-habenula; 15-foramen Monroi; 16-nucleus caudatus; 17-sulcus optostriatus et taenia semicircularis; 18-sulcus calloso-marginalis; 19-sinus corporis callosi; 20-gyrus limbicus; 21-gyrus hippocampi; 22-ligamentum Giacomini; 23-fascia dentata; 24-fasciola cinerea; 25-fimbria; 26-rudimentary gyri of Retzius. (After Testut.)

The F. has the shape of a triangle (the base of which is directed backward, and the apex forward) and is located under the corpus callosum above the optic thalami and the III ventricle of the brain, the roof of which it forms; it has two surfaces (upper and lower), three edges, and three angles (which justifies the French name for the fornix: trigone cerebral, voute a trois piliers). The upper surface of the body of the F. is strongly curved in the anterior-posterior direction; posteriorly, it merges closely with the corpus callosum, and anteriorly, it is separated from the latter by a small space. The lower surface throughout its entire extent is related to the tela chorioidea, which separates it from the optic thalami and the III ventricle. The posterior edge of the triangular plate is fused with the posterior end of the corpus callosum and participates in the formation of the splenium; the paired lateral edges are directed obliquely, are framed by the vascular plexus of the lateral ventricles, and correspond to the upper surface of the optic thalami; they participate in the formation of the foramen Monroi. The anterior angle of the F. gives rise to two bundles diverging to the sides—the anterior columns of the F. (columnae fornicis); the posterior angles are better known by the name of the posterior columns (crus fornicis); the latter columns are strongly flattened and directly fuse with the lower surface of the corpus callosum; they run obliquely backward above the pulvinar thalami optici, and then into the inferior horn of the lateral ventricle. Both posterior columns of the F. delimit a triangular space covered posteriorly by the corpus callosum; bundles of fibers arranged transversely in this space form the commissure of the hippocampus (commissura hippocampi—see below). The anterior columns of the F. are relatively massive cylindrical bundles of myelin fibers and have a comparatively more complex course. Hiding initially on each side in the regio subthalamica, the anterior columns form here the so-called covered part (pars tecta columnae fornicis, s. radix ascendens F.), then they emerge from the corpora mamillaria, forming together with the bundle of Vicq d'Azyr (tract. mamillo-thalamicus) the shape of the number 8 (Fig. 1) and are directed forward and upward, being located directly behind the anterior commissure of the brain (Fig. 2). Subsequently, the anterior columns of the F. separate partly from the lateral wall of the III ventricle and, rising upward and slightly inward, run toward each other, forming the free part (pars libera columnae F.); very soon they completely merge with each other (this fusion gives rise to the anterior angle of the body of the fornix described above). The anterior columns of the F., together with the anterior commissure of the brain, are subject to significant individual variations: for example, in most cases, these columns of the F. pass posterior to the anterior commissure

Figure 2. 1-corpus callosum; 2-ventriculus lateralis dext.; 3-fornix; 4-septum pellucidum; 5-commissura ant.; 6 and 7-infundibulum; 8-globus pallidus; 9-putamen; 10-capsula int.; 11-nucleus caudatus; 12-ventriculus lat. sin. (After Toldt.)

sometimes they double and, forming two bundles of unequal volume, enclose this commissure on both sides. The fornix is formed by fibers of two types: some are longitudinal, others are transverse. The longitudinal fibers of the fornix are more numerous and form two arcuate bundles (right and left), located on the sides of the triangular body of the fornix; posteriorly, these bundles continue into the lower crura, passing partly into the hippocampus, and partly transforming into the fimbria hippocampi, which is connected to the hippocampus and accompanies the latter into the inferior horn as far as the uncus. The general course of the anterior and posterior crura of the fornix resembles in its arrangement the shape of the letter X (Fig. 3). In the posterior angle of this X-shaped figure are located the transverse fibers, well visible from the lower surface of the body of the fornix, in the form of convex arcs, which old anatomists compared to the strings of a lyre, whence their name originated—Lyra Davidis, s. psalterium; at the present time, this system of transverse fibers of the fornix is designated as the fornix transversus, Ammon's commissure, or commissura hippocampi. Connecting both hippocampi, the fibers of Ammon's commissure are, therefore, commissural fibers, whereas the aforementioned longitudinal fibers belong to the group of ipsilateral association fibers, since they take their origin in the Ammon's horn and terminate in the lateral nucleus of the corpora mamillaria of the same side. According to Testut, not all longitudinal fibers are directed to the corpora mamillaria: at the place where the anterior commissure bends downward, the longitudinal fibers of the fornix form a bundle, described by Foville and Zuckerkandl under the name of the "olfactory bundle of the Ammon's horn." This bundle passes in front of the anterior commissure, being located between the rostrum of the corpus callosum and the substantia perforata anterior, and here it divides into two parts: one of them, with a smaller number of fibers, is directed into the olfactory nerve, the other goes as part of the diagonal band and reaches the gyrus hippocampi.

Regarding the phylogenesis of the fornix, Kappers describes the rudiments of the fornix in amphibians and reptiles, with the latter receiving its fibers from the pyramidal cells of the Ammon's horn (archicortex); these fibers are directed into the alveus and connect with the pars fimbrialis septi; here, the indicated fibers become more powerful due to the addition to them of a bundle of new fibers from the area praecommissuralis (pars septalis of the fornix); further, the fibers of the fornix bend over the anterior commissure downwards into the thalamus and are lost in the lower part of the hypothalamus—where, in mammals, the corpora mamillaria develop. In higher animals, starting from marsupials (Marsupialia), the main part of the fornix (tractus cortico-mamillaris) takes its origin from the archicortex; the fibers of this bundle are grouped into one myelin system, going from top to bottom from the anterior commissure of the brain in the direction of the mamillary bodies, where they terminate after a partial decussation (Winkler, Timmer), being reinforced on their course by fibers of the septum (Kappers). Here it must be kept in mind that the reduction of the fornix proceeds in parallel with the reduction in the volume of the corpus callosum; thus, in marsupials, in which only the genu of the corpus callosum is developed, a significant reduction of the fornix is also noted; in other lower-standing animals, along with the absence of the corpus callosum, the fornix is also absent. Ontogenetically, the fornix develops somewhat earlier than the corpus callosum. The longitudinal fibers of the fornix appear as a result of the differentiation of cellular elements and fibers of the ventral part of the marginal fold; anteriorly, these fibers fuse with the fibers of the anterior crura of the fornix, posteriorly they curve in an arc around the inner surface of the hemisphere and are separated from the thalamus only by the epithelial layer (choroid plexuses of the lateral ventricles). At the beginning of the 4th month of intrauterine life, the fibers of the fornix fuse with the fibers of the opposite side throughout their entire extent, starting from the foramen of Monro and up to the pineal gland. This fusion of the fornix occurs from front to back and gives rise in turn to the development of the septum pellucidum; the development of the fornix itself proceeds, as is known, at the expense of the terminal lamina (lamina terminalis, Kölliker). It should be kept in mind that in all kinds of agenesis of the corpus callosum, in the majority of cases, underdevelopment of the fornix is also observed (Dejerine, Forel-Onufrowicz); there are also isolated exceptions to this rule (Mingazzini). The fornix begins to be covered with myelin in the first month of extrauterine life. According to Mingazzini, the body of the fornix begins to myelinate in the third week of extrauterine life and finishes myelination in the 17th month. The fornix is a powerful centrifugal pathway of the Ammon's horn; the axons of the pyramidal cells of the latter pass into the alveus, then into the fimbria, of which the fornix is the direct continuation. The anterior end of the latter (anterior column of the fornix) behind the anterior commissure plunges into the subthalamic region and terminates, as indicated above, in the corpora mamillaria, mainly in the lateral nucleus (Sancte de Sanctis). Judging by experiments on animals, a part of the fibers of the fornix goes, however, further, forms a decussation behind the corpora mamillaria, and can be traced to the tegmentum of the pons, right up to the so-called ganglion dorsale tegmenti (Bekhterev). Some authors (Castaigne, Shipov), after destruction of the Ammon's horn in animals, found secondary degeneration of fibers going from the anterior column of the fornix along the stria medullaris to the habenular nucleus (which Kölliker had already pointed out earlier); according to the data of other authors (Vogt, O.), the degeneration of fibers extended only to the stria medullaris and could not be traced further than the anterior part of the thalamus. It is necessary to note that in animals there is also a well-developed bundle of fibers known as the "long fornix" (fornix longus). This bundle is associative, and its existence in humans was proven for the first time by Forel, and then by Kölliker, G. Elliot Smith, and described in detail in the comprehensive work of Hilpert (1921). Regarding the physiological significance of the fornix, there is still no precise and indisputable information in the literature. According to Bekhterev, "in view of the fact that the fornix arises from the apex of the Ammon's horn, which serves as the center of smell, it should be assumed that we are dealing here with a bundle transmitting to the underlying centers motor impulses arising under the influence of olfactory sensations." We find the same motor interpretation of the fornix in Flechsig. Clinical observations, which are very scarce, seem to confirm the point of view expressed by Bekhterev. The reason for the indicated inadequate information about the physiology of the fornix should be sought in the fact that in humans this formation is almost never involved in isolation in one or another pathological process; much more often it is affected together with the parts of the brain surrounding it (e.g., with the corpus callosum, with the thalamus, or with the third ventricle, etc.); with such a combination, it is extremely difficult to establish which symptom complex is due to the lesion of the fornix and which is due to the destruction of the parts surrounding it.