Odredivanje glikozilovanih proteina seruma: poredenje metoda sa tiobarbiturnom kiselinom i fenol-sumpornom kiselinom

Abstract

Određivanje sadržaja glikozilovanih proteina je važno za otkrivanje i kontrolu stanja dugotrajne hiperglikemijc u dijabetesu. Cl ovom radu određivan je sadržaj glukoze vezane za proteine seruma kod obolelih od dijabetesa, spektrolotometrijskim metodama s fenol-sumpornom kiselinom (PS) i s tiobarbiturnom kiselinom (TBA). Vrednosti vezane glukoze dobijene s PS reagensom (80,6 ± 22,8 gmol glukoze/g proteina) su bile znatno više od do-bijenih s TBA metodom (3,71 ± 1,34 pmol glukoze/g proteina). Analiza faktora, koji bi mogli dovesti do ove značajne razlike, je pokazala da su više vrednosti dobijene PS metodom posledica dodatne dehidratacije ugljenih hidrata oslobođenih u toku hidrolize glikozilovanih proteina. Do dehidratacije dolazi pri izvođenju bojene reakcije s fenol-sumpornom kiselinom. Zaključeno je da, iako se osetljivost obe metode za fruktozu kao standard ne razlikuje značajno, TBA metoda je pouzdanija od PS metode i preporučuje se za kliničku praksu.

The estimation of glycosylated proteins is useful in the detection and control of long-standing hyperglycemic conditions. In this paper the content of protein-bound glucose was determined in diabetics by spectrophotometric method with phenol-sulphuric acid (PS) and with thiobarbituric acid (TBA). The amounts of protein-bound glucose obtained with the PS reagent (80.6 ± 22.8 mmol glucose/g protein) were always considerably higher than those obtained by TBA method (3.71 ± 1.34 μmol glucose/g protein). The analysis was made of parameters that might have led to such significant difference. It was shown that in the reaction with PS reagent there was an additional dehydration of carbohydrate released during the hydrolysis of glycosylated proteins. The additional dehydration was due to concentrated sulphuric acid which at higher temperature influenced chromogen formation. On the basis of the results obtained it was concluded that, although the sensitivity of the both methods for standard fructose was comparable, TBA method was more reliable than PS method. Therefore, it is recommended in clinical practice.