A SIMPLE AUTOMATED CITRATE DETERMINATION METHOD IN URINE

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A SIMPLE AUTOMATED CITRATE DETERMINATION METHOD
IN URINE

• Dr. Berkay ÇATALOGLU
• GATA Haydarpasa Training Hospital Department of
  Biochemistry
• Istanbul-Turkey

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INTRODUCTION

• In humans, citrate is both metabolized and
  excreated by the kidney.
• Presence in urine contributes to the inhibitory
  potential against crystalization of calcium salt.
• Hypocitraturia is seen in a substantial number of
  patients with calcium nephrolitiasis.

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• The analytical methods employed for the
  assay of citrate can be classified in to four
  main groups
• Gravimetric and volumetric methods,
• Spectrophotometry,
• Enzymatic analysis,
• Chromotographic determinations (HPLC, gas
  chromotography).

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• We propose a new automated simple citrate method
  in urine.
• The principle of the method rests on the
  quantification of the yellow complex formed with
  iron chloride III and the urinary citric acid,
  measurable at 390 nm in acidic medium
• (Millan et al., Determination of citrate in
  urine by simple direct photometry Tech Brief
  Clin Chem.1987 33 1259-1260).

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Materials and Methods
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Reagents

• R1 Glycine buffer 0.5 M, pH 2
• R2 Ferric chloride (FeCl3.2H2O) (18 mmol/L)
• Concentrated ammonium hydroxide (NH4OH)
• Magnesium chloride (MgCl2) (0,2 mol/L)
• Hydrochloric acid (HCL) pH2
• Citrate standard (10 mmol/L)

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Analytical procedure

• 4 mL sample
• 0.1 mL concentrated amonium
• hydroxide
• 0.9 mL magnesium chloride
• Centrifuge

• 4 mL sample
• 0.1 mL concentrated ammonium
• hydroxide
• 0.9 mL magnesium chloride
• Centrifuge
• Adjust pH to 2, 10 M HCl
• 0,25 mL urine
• 4.5 mL HCl

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Beckman Coulter LX-20 autoanalyser was calibrated
with distillate water and 5 mmol/L standard.
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Comparison procedure

• We carried out a comparative study between our
  method and enzymatic citrate lyase method in spot
  urine (n100)
• (Petrarulo et al., Citrate in urine
  determined with a new citrate lyase method Clin
  Chem.1995 41 1518-11521).
• Citrate lyase, converts citrate in to oxalo
  acetate, which, in the presence of
  phenylhydrazine, is transformed into the
  corresponding phenylhydrazone. The
  ultraviolet-absorbing product is determined by
  absorbance measurement at 330 nm.

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RESULTS

• The evaluation of the modified method showed a
  good precision ( CV lt 3,2 intraassay CV lt 3,9
  interassay CV lt5,15).
• The method is linear between 0.05 and 15 mmol/L.
• The method was well correlated with enzymatic
  method (r0.927 plt0.001 n100 ).

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• OUR AUTOMATED MANUAL
  OTHER
• METHOD METHOD
  METHODS
• Economic Economic
  Expensive
• Easy Easy
  Requeres lab.
• 
  experience
• Including only Long time
  Long time
• one manual
  
• step (fast)
• Sensitivity Low
  Good
• good
• 
  

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CONCLUSION

• This modified, simple, fast, inexpensive and
  easily automatizable technique, seems to be
  reliable and especially more sensitive, adapting
  particularly to the detection of subjects
  suffering a deficit of citrate secretion, one of
  most significant inhibiting agent of lithiasis
  kidney formation.