Semiautomated Method for Determination of Cystine Concentration in Polymorphonuclear Leukocytes

[摘要] Cystinosis is an autosomal recessive disease caused by impaired transport of cystine across lysosomal membranes. The subsequent lysosomal storage of the poorly soluble cystine produces crystal formation and cellular damage in many tissues. The earliest involvement occurs in the renal tubules and causes Fanconi syndrome, with polyuria, dehydration, acidosis, rickets, and failure to thrive. In untreated cystinosis, the progression of renal glomerular dysfunction leads to uremia and death by 9–10 years of age unless dialysis or renal transplantation is initiated (1). The therapy of nephropathic cystinosis involves treatment with the cystine-depleting agent cysteamine (2).The most direct diagnostic method for cystinosis is measurement of leukocyte cystine content by an Escherichia coli cystine-binding protein assay. This assay is time-consuming and involves competition between nonradioactive cystine and [14C]cystine for a cystine-binding protein. The protein-bound radioactivity is then trapped on nitrocellulose filters and is inversely correlated to nonradioactive cystine (3).Here we present a method for measuring the cystine content of polymorphonuclear (PMN) leukocytes that is based on HPLC with fluorescence detection (FD) (4)(5); this method is reproducible, sensitive, and requires no radioactive reagents.Blood (4.5 mL) was drawn by venipuncture and was collected into a heparin-containing tube. PMN leukocyte separations were carried out as described by Smolin et al. (6). After sonication of the PMN leukocytes three times for 2 s (each) in 0.1 mL of 0.1 mol/L phosphate buffer, pH 7.2, containing 5 mmol/L N -ethylmaleimide (NEM), 50 μL of 120 g/L sulfosalicylic acid was added, and the cystine content in the acid-soluble fraction was determined. The protein pellet was then dissolved in 150 μL of 0.1 mol/L NaOH, and the protein concentration was …

[学科分类] 过敏症与临床免疫学