In the course of studies on lactescence of serum, it was found that the insoluble lipids displaced serum water to a degree which was occasionally great enough to be of considerable practical importance. The immediate consequence of this displacement was the finding of spuriously low concentrations of water-soluble components of serum, although their concentrations were normal when repeated on sera from which the insoluble lipids had been removed by ultracentrifugation.These observations clearly showed the need for a simple, rapid method for determination of serum water that would be applicable to sera with a high concentration of lipids. The method using Karl Fischer reagent (1) (3).previously described (7). The original and subnatant fluids were subjected to analysis.C. Gravimetric estimation of serum water:The water was evaporated and the weight loss used to calculate the serum water in grams per 100 ml. of serum (8). D. Calculation of serum water from serum protein:Serum water was calculated from the following formula, the derivation of which is explained elsewhere (8): Ws = 98.5 -0.745 P. in which W. = serum water in gm. per 100 ml. of serum, and P. = serum protein in gm. per 100 ml. E. Osmometric method for estimation of serum water:Theory: The osmotic pressure of serum is determined before and after the addition of a known amount of dry sodium chloride designed approximately to double the osmolarity of serum. The only change taking place upon this addition is an increase in the molal concentration of sodium chloride. Since the osmotic pressure of a solution is determined by the molal concentration of solute particles, the exact molal concentration of the added sodium chloride can be calculated from the increase it causes in the osmotic pressure of serum. Knowing both the absolute amount and the molal concentration of the added salt, the volume of water in which it was dissolved can then be calculated.A freezing point osmometer was used for the determination of osmotic pressure. Although osmotic pressure is not actually measured, advantage is taken of the proportionality between osmotic pressure and freezing point depression in such a way that the results are read directly as milliosmols per liter.Reagents: 1. 0.161 molar sodium chloride solution. Dilute 9.404 gm. dry sodium chloride (reagent grade) to one liter. Double distilled water.Special equipment: A Fiske Associates freezing-point osmometer was used for the determinations of osmolarity. Procedure: To prepare the dry sodium chloride exactly 2 ml. of the sodium chloride solution were pipetted into test tubes to be used later in the osmometer. These were brought to dryness in an oven at 980 C. and subsequently stored in a desiccator until ready for use. The addition of exactly 2 ml. of double-distilled water to the dry salt results in a 0.161 molal solution of sodium chlo-1483

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Since the discovery that ketone acids are produced in the body and accumulate in the blood to excess in severe diabetes, general opinion has held that the accumulation of these chemical compounds is responsible for the syndrome known as diabetic coma. The general application of the term acidosis to the condition is in itself sufficient evidence of the importance which is attached to this disorder of metabolism. Difference of opinion seems to have been restricted chiefly to the question of the relative parts played by the ketone bodies as such and by the reduction of blood alkali and diminution of pH which they caused. With the appearance and application of accurate and practical methods for the determination of blood bicarbonate and pH it has become increasingly apparent that alkali deficits of the magnitude found in diabetic acidosis, when they are produced experimentally or occur in the course of other diseases, are not necessarliy attended by a syndrome resembling that of diabetic acidosis. This has, perhaps, given more weight to the arguments of those who would hold that acetone and diacetic acid, by their anesthetic and poisonous effects, are responsible for the symptoms and fatalities. On the other hand there is but the scantiest positive quantitative evidence to support such a theory. The anesthetic actions of acetone and diacetic acid are notoriously slight, but hard to ascertain with certainty because of the ease with which normal animals excrete or oxidize these compounds. Ketosis unassociated with the other metabolic disorders of diabetes never attains so great an intensity. The most convenient experimental animals when rendered diabetic by pancreatectomy or phlorizin do not develop ketosis comparable in severity to that seen in humans with diabetic coma. Chemical analyses have demonstrated no exact correlation between the concentration of ketones in the blood and the profundity of coma in diabetic patients (20).

Previous work has demonstrated that ascorbic acid, which has been added to defibrinated blood, is taken up by the cells (1). The advantages offered by the method of Mindlin and Butler (2) have made it possible to investigate the factors which influence the passage of ascorbic acid into the cells. This paper deals with the effect of time, temperature and oxygen. METHODSAscorbi acid in serum was determined by the method of Mindlin and Butler (2) with the following modifications.(1) Neither potassium oxalate nor potassium cyanide was used. An anticoagulant was not necessary because the blood was defibrinated by stirring with a glass rod. The possible erroneous influence of added KCN has been demonstrated by other observers (3,4,5), as well as in this laboratory. Furthermore, addition of oxalate, cyanide or any other salt seemed undesirable, since changes in cell volume had to be avoided.(2) The strength of the metaphosphoric acid solution is of great significance for the reliability of the colorimetric method. With ascorbic acid a stable color develops; other reducing substances cause progressive fading of the indicator. Mindlin and Butler pointed out, and our own observations agree that, with solutions of pure ascorbic acid, the stability of the reduction of the dye depends on the pH of the final mixture of equal volumes of buffered dye and metaphosphoric acid. The dye solution invariably gave the desired pH of 7.0 when made according to the directions given by the aforementioned authors. Metaphosphoriic acid solutions, however, made up by weight from three different brands, varied widely in strength and were all weaker than theory demanded. Since it is known that metaphosphoric acid is partially converted to the ortho form on standing, the strength of a solution can be adjusted only by standardization. The metaphosphoric acid was, therefore, titrated with 0.1 N sodium hydroxide, using phenolphthalein as an indicator.In agreement with the observations of Mindlin and Butler, stable blanks were obtained when the final pH of the dye-metaphosphoric acid mixture was kept at 4.2 to 4.3. This resulted with solutions of the acid which were 0.21 to 0.22 N. The same normality was attained in 1This work was aided by a grant from the Markle Foundation.filtrates from serum when the deproteinization had been carried out with a solution of this acid 0.51 and 0.52 N.(3) Procedure. In most of the experiments large amounts of ascorbic acid were added to the blood. Smaller aliquots of serum were therefore necessary. In control experiments identical results were obtained on filtrates derived from 0.5 cc., 1, cc. or 2 cc. of serum, provided proportional volumes of distilled water and metaphosphoric acid were used in the precipitation. When 0.5 cc. was used the precipitate was thrown down by centrifuging to insure 1 cc. of filtrate for analysis. This was then diluted to 6 cc. with 0.21 N metaphosphoric acid. Four cc. of this diluted filtrate added to an equal volume of the buffered dye solution were used for the colorimetric reading.2D...

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