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and transmitted illumination. With the proper combination of lighting, the reaction spots for sulfuric acid appear as milky yellow, perfectly circular areas on a blue background. Most other acids will give yellow spots, but these will not contain the milky precipitate of barium sulfate.Phosphoric acid will also cause a milky precipitate within the reaction area; however, the barium phosphate formed is birefringent, in contrast to barium sulfate. The two can therefore be distinguished by means of crossed polars. Other sulfates and phosphates will give circular milky areas, but for the most part were not found to be sufficiently acidic to change the color of bromophenol blue.It was difficult to reproduce precisely the relationship between sulfuric acid droplet diameter and reaction spot di-ameter, presumably because of the sensitivity of the sulfuric acid droplet diameter to relative humidity. However, as a result of a large number of experiments, it is believed that the equation Dh = 2.5 Dd where Dh is the diameter of the reaction spot or "halo" and Du is the equivalent diameter of a droplet of pure sulfuric acid, represents the droplet size within ±30%. The equation is not valid above approximately 90% relative humidity. At extremely high humidities, the test fails because of the extreme dilution of the sulfuric acid drops.

CHRONOSIS is the deposition of a brown-0 black pigment in the connective tissues of patients with alcaptonuria. The essential feature of the alcaptonuria syndrome is the excess accumulation of homogentisic acid (HGA), resulting from a deficiency of the enzyme HGA oxidase, which, if present, would metabolize the HGA to maleylacetoacetic acid.l Ochronosis must then be the result of an alternate pathway of HGA metabolism, which ultimately is4 terminated as a melaninlike pigment, irreversibly bound to connective tissue components. Available data indicate that the production of this pigment probably involves the oxidation of HGA to benzoquinoneacetic acid (BQA) prior to the production of the polymerized product.2 Homogentisic acid or BQA, when injected intraperitoneally into guinea pigs, has a high affinity for albumin, skin, and ~artilage.~ However, BQA reacts chemically with proteins as contrasted to the physical binding of HGA with protein^.^ The investigation described in this paper presents an addition reaction that takes place between BQA and amino acids or other biological amines. Spectrophotometric and electrophoretic evidence obtained indicates that stable adducts were formed between the amines and BQA. The possible significance of this reaction in relation to the development of ochronosis is discussed. MATERIALS AND METHODSAdducts of BQA and biological amines were prepared for absorption studies by adding 5.0 ml. of a solution containing 5 X 10-5 moles of HGA to 10 ml. of a solution containing 20 x 10-j moles of the amine in a small beaker containing a miniature magnetic stirring bar. The beaker was then placed on a magnetic stirrer, and a set of calomel glass electrodes was inserted into the solution connected to a Beckman model G pH meter. Dilute sodium hydroxide was added to obtain the desired pH. Unless stated otherwise, a pH of 8.0 was used to obtain the oxidation of HGA to BQA. Following the pH adjustment, the solution was stirred for 30 minutes and then diluted to 25 ml. with water. The reaction time was limited to 30 minutes so as to avoid interference by compounds other than BQA which could be formed in this mixture by a longer period of reaction and to have BQA as the primary quinone in the mixture. The absorbance of this solution was determined between 600 and 320 mp. This solution was further diluted 1:25 with water, and the absorption in the ultraviolet range was obtained. All spectrophotometric measurements were performed on a Beckman Model DU Spectrophotometer using 1.0 cm. Silica cells.Samples for electrophoresis studies were prepared by taking 2 X 10-4 moles of the amine and 1 X 10-4 moles of the HGA and dissolving them in 1.0 ml. of 5% sodium bicarbonate contained in a 15 X 125 mm. test tube. These solutions were mixed several times during a 30 minute interval on a Vortex mixer. Solutions containing the HGA alone and the corresponding amine alone were also prepared in an identical manner. Samples of each were spotted in the center of a 6 X 12 inch Whatman #42 filter paper, which had beeh s...

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A relatively simple method for the quantitative determination of homogentisic acid in urine is presented. Homogentisic acid (2,5-dihydroxyphenylacetic acid) is oxidized by atmospheric oxygen in mild alkali to form 1,4-benzoquinone-2-acetic acid. The latter compound is then conjugated with 2,4-dinitrophenylhydrazine. The resulting hydrazone, in the presence of alcoholic sodium hydroxide, produces a characteristic lavender color with an absorption maximum at 570-580 mµ. A sample blank is run without oxidation to compensate for the carbonyls that may be present in the sample. The absorption curve produced is characteristic of homogentisic acid and may thus be used for its identification.