A modification of the benzidine method for measurement of hemoglobin in plasma and urine.

I x VARIOUS pathologic consdit-iomss, but especially its hemisolytic disease, it is of imiterest amid importamice to miseasure accurately the conscenstrations of dissolved hemoglobins ins the untie amid plasma. This is easy to do whens the conscenstratioms of Hb is high so that standard mnethods of hemisoglohimsonsetry4 cans be adapted to the purpose. With low conscenstrationss or smuall volumnes of mssaterial itis miecessary to amsiphify the photonsetric quality of the pigment. This msiay be domse variously. Flinsk amid Watsonsb proposed the use of pyridine, but this nsethod is niot accurate for dilute HI) solut-ioiss. The benzidine reactions has beets the most widely used. It was first proposed by Wu’#{176}as a misicronisethod for liensoglobimsomisetry amid, although the nsethod has beets repeatedly misodifled,’ . . 7-9 it has not proved to he ams accurat-e method for Isseasurinig hensoglobini ins the plasnsia2 or the urimse.6 We have studied the bemszidimse method amid have foumid ins plasnssa amid mmrinse several sources of interference with the color developnsienst of the benszidiniehensochromisogens reactions. In plasma there is ans initerfering stmbstamsce, perhaps lipoproteimi, that cats be dissolved ins a mixture of nssethamsol and chloroform. The additions of alcohol to the reactions, as proposed by Harms,7 inisproves the recovery frons plasmisa, hut the recovery is not complete and it varies wit-h the ansounit of plasnisa that is used, a source of imscomsveniensce whets it is niecessary to dilute the unkmiowms ins order to brimig it withims the ransge of the miset-hod. Alcohol also causes darkening of most bemszidinie solutions so that it is usually niecessary to nsake the reagemst fresh each t-imue it is to be used onto add the alcohol to each reactioms after the hensioglobins and I)emizidinse solutions have beets nsixed. We have foulsd this latter expediemit a useful one. Ins our clinical studies for the past two yean-s, we have used a misodifications of Ham’s misethod, inixinig 0.02 nil. of the uniknsow’ns with 2 ml. of a 2 per (-emit solutions of benzidinse dihydrochloride ins 20 per censt acetic acid amid then addimsg 1 isil. of 95 per cemst ethansol before the additions of 1 miii. of 0.5 per censt hydrogens peroxide. Evens wit-h this method the recoven’y of hemssoglohims ins the presence of plasma may be deficieist by as much as 10 to I 5 per (-emit (table i). Urine comitains urates, sulfates ansd perhaps other misatemials that form inssoluble complexes with benzidinse and iiot onily insterfere wit-h color developmenst but also cause turbidity. Sinsce the degree of insterferensce is proport-ionsal to the conscenstrat-ions of these nsaterials it is msot satisfactony to balansce the react-ions of a um-inmary unsknsowns by addimsg a sinisilar ansounst of nsornsal urinse to the knsowns standan-d hennoglohims solutions.6