On the Chemistry of Hammarsten Test for Indican in Urine

Physiological indican, indoxyl sulphate, is an analyte since higher levels of this compound indicate stomach dysfunction and intestinal dysbiosis. The Hammarsten test for indican in urine is interesting from the Biochemistry point of view as for Organic Chemistry since the chemistry involved in this test has not been described. The test employs a solution of calcium chlorohypochlorite added to acidulated urine with fuming hydrochloric acid, in the presence of chloroform. The latter turns blue due to indigo formation. The colour can be pale, bright, or dark, in accordance with indican concentration. This swift indigo-synthesis in acidic medium, via chlorinated intermediates, differs notably from the oxygenation process in alkaline medium that takes place during the obtention of indigo from vegetable source. We provide the route from physiological indican to indigo blue and to indigo red (indirubin), giving the electron flow, step by step, in this reaction series.