Urinary Glycosaminoglycans Determination, by Colorimetric Method using DMB (Dimethylmethylene Blue) in Alkaline Solution, in Healthy Dogs
*Luis Octavio Lopez Vazquez, Marcia Mery Kogika, Marcio Dentello Lustoza, Mitika Kuribayashi Hagiwara, Regina M. Sakata Mirandola
*Faculdade de Medicina Veterinaria e Zootecnia da Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87
São Paulo, BR
Glycosaminoglycans (GAGs) are mucopolysaccharides (heparan, dermatan and chondroitin sulphates and hyaluronic acid), which can be found in different tissues of the body. In urinary tract, GAGs allow the formation of a layer of water/urine in the surface of the mucosa, protecting and also facilitating the elimination of bacteria during micturition. In women and cats, alteration of GAGs layer as well as reduction of urinary excretion of GAGs is mentioned in interstitial cystitis (IC). In dogs, low concentrations of urinary GAGs seem to be associated with recurrence of urinary infections and urolithiasis. Disorders of GAGs degradation and presence of large amount in urine can be observed in mucopolysaccharidosis. Most of GAGs laboratorial colorimetric methods reported develop in acid pH solution with DMB (colorant), but in this condition, DMB can also react with other urinary proteins. Jong (1992) mentioned a modified method, in which DMB reacts only with GAGs, forming a steady complex, and this reaction develops in alkaline solution. The purpose of the present study was to evaluate urinary GAGs in healthy dogs, by using of colorimetric method of DMB in alkaline pH solution.
Thirty-two healthy dogs of different breeds or mongrel were composed in 2 groups: 16 dogs (1 to 3 years), divided in 8 males and 8 females, and another group of 16 adult dogs (4 to 6 years), also distributed in 8 males and 8 females. Urinary GAGs determination was performed as described by Jong (1992); a steady complex of the colorant (DMB) with GAGs is formed in alkaline pH solution, and then, there is no interference of other urinary proteins in total GAGs measurement. Standard GAGs solution was used for determination of standard curve, and a volume of 50 µL of urine sample was necessary. The absorbancy was measured in espectrophotometer at 520 nm. Results was expressed in [mg of GAG/mmoles of urinary creatinine], unit that eliminates the interference of urinary specific gravity.
GAGs measurements, observed in 32 urine samples of healthy dogs, presented values of 2.30 ± 2.01 mg of GAG/mmoles of urinary creatinine (mean ± standard deviation); standard error of mean was of 0.36; median was 1.77; maximum value observed was 7.61 and minimum zero.
Considering that no normal values for urinary GAGs in healthy dogs was described in the literature, total urinary GAGs concentration values observed in normal dogs in the present study could help in further studies, in order to compare these values with those to be observed in different morbid processes that involves alteration in GAGs metabolism. Furthermore, the use of DMB in alkaline solution reaction, an easy execution method, that assures the development of a stable complex between GAGs and DMB, besides there is no interference of other urinary proteins in total GAGs measurement, this method can be considered reliable for routine procedures.