Urinary & serum oxalate determination by oxalate oxidase immobilized on to affixed arylamine glass beads.

BACKGROUND & OBJECTIVE: Although the measurement of oxalate in urine and serum by Amaranthus leaf oxalate oxidase immobilized on free arylamine glass beads is highly sensitive and specific, the handling of glass beads is tedious and cumbersome. The present study was undertaken to overcome this problem. METHODS: Partially purified Amaranthus spinosus leaf oxalate oxidase was immobilized through diazotization onto arylamine glass beads affixed on the surface of a plastic strip by a non reactive fixative and employed for oxalate determination in urine and serum samples collected from healthy individuals and urinary stone formers. RESULTS: The immobilized enzyme retained 56 per cent of its initial activity with a conjugation yield of 40 mg/g support. The strip bound enzyme showed maximum activity at pH 3.5 when incubated at 40 degrees C for 15 min. The minimum detection limit of the method was 0.01 mM/l in the urine and 2.5 microM/l in the serum. The analytical recovery of added oxalate was 97.7+/-1.2 per cent in urine and 92.0+/-2.4 per cent in serum. Within and between assay coefficient of variation (CV) were 4.6 and 5.2 per cent in urine and 7.4 and 5.8 per cent in serum respectively. A good correlation for oxalate in urine (r1= 0.99) and in serum (r2= 0.92) was obtained between Sigma kit method and the present method. The strip could be reused 150 times over a period of 2 months, when stored at 4 degrees C in reaction buffer. INTERPRETATION & CONCLUSION: Immobilization of Amaranthus leaf oxalate oxidase on to affixed glass beads provided enormous ease in its reuse for determination of oxalate in urinary and serum samples.

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants.

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.