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TECHNICAL NOTE
Year : 2017  |  Volume : 40  |  Issue : 1  |  Page : 44-47  

A simple method for the estimation of phosphorus in urine


Radiation Safety Systems Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India

Date of Submission17-Feb-2017
Date of Acceptance06-Mar-2017
Date of Web Publication24-Apr-2017

Correspondence Address:
Seema Chaudhary
Radiation Safety Systems Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/rpe.RPE_8_17

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  Abstract 

32P is preferentially eliminated from the body in urine and is estimated by in situ precipitation of ammonium molybdophosphate (AMP) in the urine followed by gross beta counting. The amount of AMP formed depends on the amount of stable phosphorus (P) present in the urine. Hence, the present study was undertaken to estimate daily urinary excretion of P by the spectrophotometry method. P forms a colorless complex (phosphomolybdate) with molybdic acid, which on reduction produces deep-blue-colored complex called molybdenum blue. The intensity of this blue color is directly proportional to the amount of P present in the sample. 24 h urine samples collected from radiation workers were analyzed for stable P, and its range was observed to be between 0.4 and 1.4 g/day. This information was valuable in finalizing volume of the urine sample required for analysis of 32P in bioassay sample by gross beta counting technique.

Keywords: 32P, ammonium molybdophosphate, molybdenum blue, phosphorus, spectrophotometry, urine


How to cite this article:
Chaudhary S, Gondane S, Sawant PD, Rao D D. A simple method for the estimation of phosphorus in urine. Radiat Prot Environ 2017;40:44-7

How to cite this URL:
Chaudhary S, Gondane S, Sawant PD, Rao D D. A simple method for the estimation of phosphorus in urine. Radiat Prot Environ [serial online] 2017 [cited 2018 May 27];40:44-7. Available from: http://www.rpe.org.in/text.asp?2017/40/1/44/205057


  Introduction Top


Following internal contamination of 32 P, it is preferentially eliminated from the body in urine [1] and is estimated by in situ precipitation of ammonium molybdophosphate (AMP) in the urine followed by gross beta counting. The amount of AMP formed in situ depends on the amount of stable phosphorus (P) present in the urine. If concentration of stable P in the urine is significant, then the amount of AMP formed would also be higher, leading to absorption of some of the β particles emitted by 32 P [Figure 1]. The present study was undertaken to estimate daily urinary excretion of P to finalize the volume of urine to reduce absorption of 32 P beta by AMP precipitate formed in situ.P present in the urine interacts with molybdic acid to form colorless phosphomolybdate, which on reduction forms a deep blue-colored complex called molybdenum blue. The reducing agent used in the present study was p-phenylenediamine dihydrochloride.[2] The amount of the molybdenum blue formed in the sample is directly proportional to the amount of P present in the sample. Thus, daily urinary excretion ofPwas estimated in 24 h urine samples of radiation workers, and based on the observed range, volume of the sample required for 32P estimation in bioassay samples was finalized.
Figure 1: 32P activity (21 Bq) measured with correction and without correction for self-absorption by ammonium molybdophosphate precipitate

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  Materials and Methods Top


Phosphorus standards

80 ppm P standard was prepared by dissolving 35.15 mg of potassium phosphate monobasic (AR grade, make Chemie Centre) in 100 mL of distilled water. Other standards of P (2.4, 5, 10, 20, and 40 ppm) were prepared by further dilution of 80 ppm standard with distilled water and used for calibration of the spectrophotometer.

80 ppm P standard was used for optimizing the various parameters and concentration of reagents required for spectrophotometric analysis of stable P.

During the standardization, duration required for stable blue color development was also studied from 5 min onward up to 24 h.

Sulfuric acid solution

1–18 N H2 SO4 solutions were prepared by diluting concentrated H2 SO4(AR grade, 98%, make Thomas Baker) with distilled water.

Molybdic-trichloroacetic acid reagent

Molybdic acid solution

0.5–5.0 g of ammonium molybdate (AR grade, make J. T. Baker Chemical Co., NJ, USA) was dissolved in 50 mL of 10 N sulfuric acid solution.

Trichloroacetic acid solution

10% solution was prepared by dissolving 10 g of trichloroacetic acid (TAA; LR grade, make S.D. Fine Chem. Ltd.) in 100 mL of distilled water.

Molybdic TAA (MTAA) was prepared by mixing one part molybdic acid with two parts of 10% TAA solution (1:2, v/v).

Reducing agent

0.1–1.0 g of p-phenylenediamine dihydrochloride (AR grade, make Chemie Centre) was dissolved in 5% sodium metabisulfite (AR grade, make Chemie Centre) solution.

Instruments

Spectrophotometric analyses for stable P were carried out using Elico SL-160 double-beam UV-VIS spectrophotometer.

Beta counting was carried out using a radiometer (Doza, UMF-2000).

Procedure standardized

After optimizing the conditions for color development with respect to normality of H2 SO4, concentration of MTAA as well as reducing agent, and the time required, the procedure for estimation of stable P in the urine samples was standardized [Figure 2] as follows: 1 mL of 24 h urine sample was diluted to 10 mL (1:10, v/v) with distilled water. 0.5 mL of this diluted urine sample was taken in a centrifuge tube; 0.5 mL of MTAA was then added to it. After 10 min, 0.5 mL of this solution was transferred to a quartz cuvette along with 2 mL of reducing agent and allowed to stand for 30 min for color development. For calibration of the spectrophotometer, P standards and blank sample were also prepared in a similar way. In case of blank sample, diluted urine sample was replaced by distilled water. The readings of P standards as well as urine samples were taken against blank at 700 nm.
Figure 2: Flow chart for estimation of stable phosphorus in urine

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  Results and Discussion Top


The addition of MTAA solution to diluted urine sample results in the formation of phosphomolybdate anionic complex [PMo12O40]3− which is colorless and has an α-Keggin structure. [PMo12O40]3− can accept electrons from a reducing agent to form blue-colored mixed-valence complex [PMo V4 Mo VI8O40]7− which has a β-Keggin structure. This deep-blue-colored complex is called molybdenum blue. The intensity of blue-colored complex formed is directly proportional to the amount of P present in the sample. The maximum absorbance (λmax) forPwas observed to be 700 nm [3] and the same was used in the present study.

The absorbance for molybdenum blue increased with the normality of sulfuric acid and then remained unchanged up to 18 N [Figure 3]a. Similarly, when the amount of ammonium molybdate was varied in MTAA solution from 0.5 to 5.0 g, optimum absorbance was observed when weight of ammonium molybdate was 2.5 g in MTAA solution [Figure 3]b. The experiment with reducing agent having variable amount of p-phenylenediamine dihydrochloride in 5% sodium metabisulfite solution indicated maximum absorbance of molybdenum blue at 0.5 g of p-phenylenediamine dihydrochloride. Absorbance reduced significantly at higher concentrations due to precipitate formation [Figure 3]c.
Figure 3:

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Time required for color development was studied from 5 min onward up to 24 h and it was observed that color development was completed within 25–30 min and remained stable for the next 24 h [Figure 3]d. All the absorbance measurements were, therefore, carried out at the end of 30 min of color development. Various P standards were analyzed for obtaining the calibration curve [Figure 4] as well as to study the accuracy of the method standardized. The recovery was observed to vary from ~95 to nearly 100% [Figure 5], with an average recovery of 98.2% (±3.1% standard deviation). The method was then applied to urine samples collected from 59 radiation workers to measure the daily urinary excretion of stable P [Figure 6]. The amount of stable P in 24 h urine sample was computed as follows:
Figure 4: Calibration curve for estimation of stable P in urine samples

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Figure 5: Recovery of stable phosphorus observed by the method standardized

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Figure 6: Daily urinary excretion of stable phosphorus

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Where P is daily excretion of stable P in the urine (g/day), AS is absorbance of stable P in sample, ASTD is absorbance of stable P in standard, CSTD is concentration of P standard (μg/mL), 10 is the dilution factor, V is volume of urine excreted per day (mL/day), and 106 is the conversion factor (μg to g).

The range of stable P in urine was observed to be from 0.4 to 1.4 g/day, with an average of 0.78 ± 0.23 g/day, and is comparable with the literature values: 0.4–1.3 g/day [4] and 0.3–1.0 g/day.[5] In the present study, it was observed that average urinary excretion of stable P for workers was ~0.78 g/day. The average volume of 24 h urine samples collected for the analysis was 2200 mL, which corresponds to ~0.35 mg ofPexcreted per mL of urine sample. [Figure 1] indicates that as the volume of the urine sample taken up for 32 P analysis increases, there is absorption of beta particles by AMP precipitate formed in situ during the procedure. It is also observed that minimum absorption of beta particles is at 0.5 g of AMP (<5% reduction in 32 P activity), and to obtain 0.5 g of AMP in situ, the amount ofPrequired is ~8 mg in the urine. Therefore, based on these findings, 25 mL aliquot of the urine sample was finalized for estimation of 32 P activity in urine samples.


  Conclusions Top


The method standardized in the present study is simple, easy and is successfully implemented for estimation of daily urinary excretion of stable P. The present study provided useful information on excretion of stable P in the urine. The range of stable P in urine observed in the present study was comparable with the values reported internationally. It also provided a valuable input for finalizing the volume of urine sample required for analysis of 32 P in bioassay sample by gross beta counting technique.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Dose coefficients for intakes of radionuclides by workers. A report of a Task Group of Committee 2 of the International Commission on Radiological Protection. Ann ICRP 1994;24:1-83.  Back to cited text no. 1
    
2.
Jung DH, Parekh AC. Urinary inorganic phosphorus determinations. J Clin Pathol 1972;25:263-5.  Back to cited text no. 2
    
3.
Hurst RO. The determination of nucleotide phosphorus with a stannous chloride-hydrazine sulphate reagent. Can J Biochem 1964;42:287-92.  Back to cited text no. 3
    
4.
Tournis ST, Giannikou PV, Paspati IN, Katsalira EA, Voskaki IC, Lyritis GP. Co-existence of X-linked hypophosphatemic rickets (XLH) and primary hyperparathyroidism: Case report and review of the literature. J Musculoskelet Neuronal Interact 2005;5:150-4.  Back to cited text no. 4
    
5.
Chaves EL, Hernandez-Artiga MP, Munoz-Leyva A. Spectrophotometric phosphate determination in urine by ligand exchange extraction. Mikrochim Acta 1994;116:91-9.  Back to cited text no. 5
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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