W.C. Hess

Determination of neuraminic (sialic) acid, glucose and fructose in spinal fluid☆☆☆

Abstract A procedure is described for the colorimetric determination of neuraminic acid, fructose, and glucose in spinal fluid. The amount of total neuraminic acid, as determined by the orcinol test, is calculated after due correction for fructose and glucose interference. The method has been compared with an ion-exchange method, with favorable results. The procedure is rapid, requires a very small amount of spinal fluid, and can be used routinely for the determination of total, free, and bound neuraminic acid, fructose, and glucose.

Paper chromatographic detection of sugars in normal and dystrophic human urines.

Abstract A method has been developed, involving the use of a mixed-bed resin column, for the preparation of a urine concentrate suitable for the paper chromatographic detection of small amounts of sugars. The urines of 15 normal adults and 11 children and adults afflicted with muscular dystrophy were examined. The following sugars are commonly present in the urines from normal humans: lactose, galactose, glucose, ribulose, arabinose, xylose, ribose, glucuronolactone, fructose, xylulose, fucose, sucrose, mannoheptulose, and sedoheptulose. The presence in urine of the last four sugars has not been reported previously. The same sugars were found in the urine of persons with muscular dystrophy. Under the conditions of this study, that is, without 24-hr, urine samples from the patients, there were no obvious changes from the normal range of spot sizes and intensities.

The lipide content of enamel and dentin.

Sample Preparation.The dentin used was prepared in the same manner as that previously used for the isolation of cholesterol.1 The enamel was prepared by the method employed by Losee and Hess2 for the isolation of enamel protein. Cholesterol in Enamel.-The method of isolation of cholesterol from dentin that was most satisfactory was used for the isolation and estimation of the cholesterol in enamel. The procedure involved extraction of the enamel with an aqueous solution containing 30 per cent KCl and 1 per cent K2C03. Twenty milliliters of solution was used for each gram of enamel. After several extractions the combined extracts were treated with 0.4 Gm. of potassium acetate, 0.4 Gm. of silica gel, and 0.4 ml. of glacial acetic acid per gram of original sample. The extract was then shaken for 30 minutes, filtered with suction through a fine-sintered glass filter, and the filtrate was extracted with petroleum ether. Both the petroleum ether extract and the aqueous extract were analyzed for total cholesterol. To determine if the treatment with silica gel adsorbed any cholesterol, a recovery experiment using pure cholesterol was run in which 2 solutions, each containing 1.76 mg. of cholesterol in 100 ml. of H20, were used. One solution was treated with the same amount of silica gel used in the enamel extraction procedure and the other was used as a control. Cholesterol determinations gave a value of 1.76 mg. in the control and 1.43 mg. in the silica gel-treated solution, a loss of 18 per cent. Correction for this loss incurred in the procedure was used on the determinations of the cholesterol content of the enamel. All cholesterol determinations were made by the same method previously used.1 The enamel employed weighed 8.89 Gm., the aqueous extract contained 4.4 mg. of cholesterol per 100 Gm. of enamel, the petroleum ether extract

Some alterations in serum enzymes in progressive muscular dystrophy.

Summary Acid and alkaline phosphatases, transaminase, and aldolase were determined in sera of 8 children and 9 adults afflicted with progressive muscular dystrophy. There was no alteration in phosphatase activity. Aldolase was increased in both children and adults while transaminase was distinctly elevated only in children. The ratio of aldolase to transaminase activity in serum increased in muscular dystrophy. The results indicate that increased activities of both aldolase and transaminase in progressive muscular dystrophy are derived from diseased muscle.

Isolation and determination of the glycoproteins in cerebrospinal fluid.

Summary A procedure is described for the isolation of the glycoproteins from spinal fluid that also separates the lipides and free sugars. The carbohydrates in the glycoproteins were identified by paper chromatography, mannose, galactose, fucose, and glucosamine were found. 2. Methods are given for the quantitative estimation of the hexose and hexosamine associated with the glycoproteins and the gamma globulin, and also the neuraminic acid content of the spinal fluid. 3. An increase in gamma globulin hexose was found in multiple sclerosis. The ratio of gamma globulin hexose to glycoprotein was likewise increased. However the ratio of hexose to gamma globulin was not different from that found in non-demyelinating diseases, indicating that the hexose increase merely reflected the increase in gamma globulin.

Dentinal Protein: Bound Cholesterol

EVERAL authors have mentioned that lipids are constituents of human 3 teeth. Akamatsul and Kokubun2 used Sudan III and other Sudan dyes to demonstrate this fact histochemically in ground slices of enamel. Kokubun also mentioned that fat occurred in the dentinal portion of the tooth sections with which he worked. Using a quite different approach to show the presence of lipids, Krasnow3 determined that enamel and dentin both had measurable lipid phosphorus content. Pincus4 isolated, from the organic material in enamel, about 1 per cent of a fatty substance the nature of which he did not elucidate. Pincus stated that sterols were a possible constituent which had been overlooked in teeth. The present communication deals with the presence of cholesterol as a normal constituent of the root dentin of human teeth and its extraction from the dentin.

MICRODETERMINATION OF FREE AND ESTERIFIED CHOLESTEROL IN CEREBROSPINAL FLUID

IN order to study the cholesterol levels in the cerebrospinal fluid (CSF) of patients with demyelinating and other neurologic disorders, an accurate microassay w e devised for the extraction, separation, identification and determination of free and esterified cholesterol. The micromethod is based on an extraction procedure, ROBOZ et a/., (1958), separation by silicic acid column chromatography, FILLERUP and MEAD (1953), WYCOFF and PARSONS (1957), and colorimetric determination by H,SOpFeC1, reagent, ZLATKIS, ZAK and Bow (1953), MACINTYRE and RALSTON (1954), ROSENTHAL, PFLUKE and SALVATORE, (1957). SELBACH and TWPE (1944) studied the levels of free and esterified cholesterol in CSF specimens from patients with psychiatric and neurologic diseases using an aluminium oxide column. Aluminium oxide is reported to hydrolyse esters TRAPPE (1940), FILLERUP and MEAD (1953). This difficulty was not encountered by H ~ s s (1947) in previous work in blood. Rosoz et al. (1958) used aluminium oxide to separate free and esterified cholesterol in CSF. This method requires large amounts of aluminium oxide and eluents. It was found that silicic acid could be employed for the same purpose. It has the advantage that only a small amount is required for the column and the volume of eluants is greatly reduced. No hydrolysis of esterified cholesterol was detected. The free and esterified cholesterol in CSF were identified by paper chromatography. The method has been applied to the analysis of CSF of patients with non-neurologic disorders.

Amino acid composition of dentin protein.

Summary Protein prepared from the dentin removed from the crowns of non-carious, fully erupted, permanent, first and second human molars has been analyzed for lysine, arginine, histidine, hydroxyproline, phenylalanine, methionine, cystine, tryptophan, serine, threonine, glycine, alanine, and tyrosine. The amounts found of many of these 13 amino acids, particularly the values for glycine, hydroxyproline, and the absence of tryptophan suggest that dentin protein resembles collagen.

Effect of administration of ACTH and cortisone upon blood glutathione levels.

Summary Blood glutathione (GSH) and total glutathione (GSH plus GSSG) were determined in eleven patients during ACTH therapy and in eleven patients during the administration of cortisone. GSH values were significantly decreased from a pretreatment average of 36.6 mg per 100 cc to 26.9 mg by ACTH while cortisone produced a decrease from an average pretreatment value of 35.0 mg per 100 cc to 29.1 mg. The latter decrease, statistically, is of a low order of significance. The total glutathione values showed no changes following the administration of either ACTH or cortisone. During treatment with ACTH the greatest decrease in blood GSH occurred in the patients showing the greatest increase in blood sugar. There is suggestive evidence that although patients with normal glucose tolerance may show diabetic patterns during treatment, those who demonstrate mildly diabetic patterns prior to therapy show no further decrease in glucose tolerance and minimal reduction in GSH values after ACTH or cortisone therapy.

Studies in Cancer The Application of the Rupp-Schied-Thiel Thiocyanate Reaction to the Urine.

Saxl 1 concluded that quantitatively thiocyanate is increased in cancer with values appreciably higher than normal and higher than found in any other disease. He employed the Rupp-Schied 2 method as improved by Thiel. 3 Making use of the same method we found great variation in the apparent thiocyanate content of both normal and pathological 24-hour urines. A number of cases of marked cancer involvement showed high apparent thiocyanate as found by Saxl but other marked cases of cancer were within normal limits and sometimes below normal. It would seem that the excretion of material behaving like thiocyanate is not necessarily increased in cancer. The average in cancer is somewhat higher than for other pathological conditions tested by us since some cases as, for example, multiple myeloma, were much higher than normal. The values found in our cancer cases show that irrespective of what the Rupp-Schied-Thiel thiocyanate procedure is measuring when applied to urine, thiocyanate or other similarly reacting material is not necessarily increased in cancer.