8/2/2019 Inhibition of the In Do Phenol Reaction in the Spectrophotometric Determination of Ammonia

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CLINICA CHIMICA ACTA 59

INHIBITION OF THE INDOPH ENOL REACTION IN THE SPECTROPHOTO-METRIC DETERMINATION OF AMMONIA

SUMMARY

The development of the indop henol colour formed by amm onia, ph enol andhyp ochlorite was inhibited by Tris, glutamine and ph osphate. These comp ound s d idnot alter the indop henol colour after its development.

The blue colour of indop henol formed by amm onia, ph enol and hyp ochloritein an alkaline med ium was first described by Berthelotl, and is now utilized by manywor kers in the assay of ammonia. Preliminary to a stud y of glutam inase activity indog kidney, I attempted to adap t th is method to measure the rate of amm onia pro-du ction from glutamine. The samp les consisted of sup ernatants from dog kidneyhom ogenates, glu tamine, tris (hydr oxymethy l)methylamine (Tris) and ph osph ate.The results obtained by this method were only 20 to 25% of those found with theConw ay microdiffusion technique. I then investigated this d isparity.M E T H O D S

Solution IPhenol (50 g) and sodiu m n itropru sside (250 mg) dissolved in I liter of water.

Solutio9r 2Sodium hyd roxide (24 g) and sodium hyp ochlorite (2.1 g) dissolved in I liter of

water.Two ml of solution r were ad ded to I ml of samp le and mixed. Then 2 ml of

solution 2 were add ed, mixed and allowed to stand for I h at room temperatu re. Thecolour developed was read in a Zeiss mod el PM Q II spectroph otometer at a wav elength of 630 nm . With stand ard NH ,Cl samples there w as a straight line relationshipbetween absorban ce and concentration up to a valu e of 0.7 ,uequ iv NH &l. This pr o-cedu re is similar to that described by Chaney and Marbacha.

Pr esent adclress: D. J . ODonovan , Ph ysiology Depar tm ent , Univers ity College, Galway, Irelan d.Clin. Cizirn. Acta. 32 (1971) 59-61

8/2/2019 Inhibition of the In Do Phenol Reaction in the Spectrophotometric Determination of Ammonia

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8/2/2019 Inhibition of the In Do Phenol Reaction in the Spectrophotometric Determination of Ammonia

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AMMONIA SPECTROPHOTOMETRY 6 1

hom ogenate gave a value of 0.5 pequ iv NH ,, wh ich should have given an absorbanceread ing of 0.520. Furtherm ore, I ml of this sample gave a lower absorbance readingthan 0.5 ml diluted to I ml with water.

Then glutam ine, ph osphate and Tris were incorporated separately in standardSH,Cl solutions at the same level as they w ere add ed to the hom ogenizing fluid.It can be seen in Fig. z that each of these compou nd s inhibit the colour developm entby ammon ia. The inhibition was greatest by Tris, bu t glutam ine is also a potent inhib-itor.

&%en th e volum e of ph enol an d hyp ochlorite were increased in the presence ofthe inhibitors, there w as only a slight rise in the absorban ce read ings. Even w ith theadd ition of 15 ml each of ph enol and hyp ochlorite the absorban ce read ings wer e stilllower than that of a standard NH &l solution withou t inhibitors.

The inhibition of colour development by Tris and glutamine was similar whenadd ed before or after the ph enol solution. How ever, wh en these comp ound s were add edafter ph enol plus hyp ochlorite the colour developed by ammon ia was not inhibited.BYlen the order of add ition of ph enol and hyp ochlorite was reversed, glutam ine andTris inhibited colour development wh en add ed after hyp ochlorite, but not after hypo-chlorite plus ph enol.

Glutam ine only slightly inhibited colour development wh en add ed at the sameconcentration as it occur s in blood. However, Tris caused a d rastic red uction inabsorban ce readings even at concentrations below wh ich it could act as an effectivebuffer, c.g. 0.001 M.

It is obvious that wh en Tris is used as a buffer in biological fluid s, the ind oph enolreaction can not be emp loyed in the determination of amm onia withou t prior micro-diffusion.ACKSOWLEDGEMENT

This investigation was sup por ted in part by a Public Health Service Inter-national Postd octoral Research Fellowsh ip (num ber 1F 05TWO 1402-01Ar).IIEFEKENCESI Al. P. E. RERTHELOT. h'epevtoiree Chimle appliquee, I (1859) 284.2 E. J. CONWAY, Microdiffus ion Ana lys is and Volum etvic Errov, McMillan, New York , 195X3 X. L. CHAWEY and E. P. MARBACH, Clin. Chem., 8 (1962) 130.

Clin. Chim. Acta, 32 (1971) 59-61