An ion-based chromogenic method of detecting for inorganic phosphate in serum and milk

doi:10.4236/health.2009.12013

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Vol.1, No.2, 76-82 (2009)

doi:10.4236/health.2009.12013

SciRes

Health

An ion-based chromogenic method of detecting for

inorganic phosphate in serum and milk

Cai-Xia Yin1*, Jing Su1, Fang-Jun Huo2*, Pin Yang1

1Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi

University, Taiyuan, China; yincx@sxu.edu.cn

2Research Institute of Applied Chemistry, Shanxi University, Taiyuan, China; huofj@sxu.edu.cn

Received 10 July 2009; revised 21 July 2009; accepted 22 July 2009.

ABSTRACT

A rapid method for the determination of inor-

ganic phosphate in serum and milk by an

ion-based chromogenic is described. Serum

samples were detected directly by our system,

and milk was also detected after degreased

through centrifugation. By this procedure the

samples are not diluted. Mean serum inorganic

phosphate concentration found in healthy indi-

vidual is 1.14mmol/L. Values found in serum is

in good agreement with those previously re-

ported. Mean inorganic phosphate concentra-

tion from foremilk and commercial milk are

2.5mmol/L and 12.5mmol/L respectively.

Keywords: UV-Vis Spectra; Pyrocatechol Violet;

Ytterbium Chloride; Phosphate; Serum; Milk

1. INTRODUCTION

Phosphate is involved in important biomineralization

processes such as bone formation and also processes that

are clearly pathological such as the genesis of renal

stones. Consequently, its determination in biological

fluids is important [1]. In a clinical setting, inorganic

phosphate levels in serum are determined as part of a

routine blood analysis. The typical inorganic phosphate

concentration in human serum range is 0.81-2.26mmol/L

[2]. Individuals with abnormally high phosphate levels

are diagnosed with hyperphosphatemia, which manifests

in acute or chronic renal failure, hypoparathyroidism and

excessive Vitamin D intake. And we know that higher

serum phosphate levels would be associated with in-

creased mortality risk among people with CKD [3-5].

Those with low inorganic phosphate levels suffer from

hypophosphatemia which can be associated with rickets,

hyperthyroidism, or Fanoci Syndrome [6-8]. In addition,

there is generally a reciprocal relationship between se-

rum calcium and inorganic phosphorus levels. High in-

organic phosphorus in serum restrains the intake of cal-

cium.

Regarding the newborn baby, foremilk or milk is the

most main headspring they grow on. But exactly this

time is the quickest time when young child grows, being

in the bone blooming period and the cerebrum and the

intelligence still being imperfect stage. So the right

amount nutrition could guarantee the normal growth, and

prevent malnutrition, rickets, anemia and so on. Espe-

cially if absorbing calcium phosphorus imbalance, can

cause the low calcium blood sickness, the rickets [9].

Moreover iron in the milk is easy to form insolubly iron

compound when affected by high phosphate and calcium,

cannot be absorbed by the human body, which may

cause the young child to occur lacking the iron anemia.

In normal foremilk the calcium phosphorus proportion is

2: 1, is easy to be absorbed, to prevent and control the

rickets. But the milk is 1: 2, is not easy to be absorbed.

Therefore, determination the calcium phosphorus con-

tent from foremilk and milk is very important.

Most of the procedure for the colorimetric determina-

tion of inorganic phosphate are based on the formation

of molybdophosphoric acid with further reduction to

heteropolymetric molybdenum blue [10-12] on direct

measurements of molybdo- and vanadomolybdophos-

phoric acid [13,14], or on complex formation between

molybdophosphoric acid and basic dyes [15]. These

chemical methods have serious shortcomings, however:

Molybdate reduction is affected by slight changes in pH,

the rate of complex formation is markedly influenced by

protein concentration, and the acidity required leads to

hydrolysis of organic phosphate, which results in over

estimates of Pi concentration [16].

In our work, we developed a rapid method for the de-

termination of phosphate in serum and milk by an

ion-based chromogenic. Serum samples were detected

directly by our system, and milk was also detected after

degreased through centrifugation. By this procedure all

samples are not diluted. Owing to our system’s promi-

nent advantages, it can serve as a hopeful substitute for

C. X. Yin et al. / HEALTH 1 (2009) 76-82

Openly accessible at

the molybdenum reagent.

2. MATERIAL AND METHODS

2.1. Reagents and Chemicals

The chemicals used were of analytical-reagent grade. PV

(Pyrocatechol violet) was purchased from Shanghai and

sodium monhydrogenphosphate was purchased from

Beijing. Ytterbium oxide was a product of Rare Earth

Graduate School of China. HEPES was purchased from

Sigma. All solutions were made up with deionized water.

HEPES buffer solutions were obtained by adding NaOH

0.1M solution into 10 mM aqueous HEPES using a

Beckman Φ50 pH meter. Ytterbium chloride was pre-

pared from ytterbium oxide and 37% hydrochloric acid.

Ytterbium ion solution was prepared by dissolving ytter-

bium chloride in water. Serum samples were collected

from health volunteers and stored at -17℃ until ana-

lyzed. Cow serum was purchased from commercial

pured product. Human milk and commercial milk were

degreased through a Centrifugal filter.

2.2. Instruments and Apparatus

pH determinations were performed using a Beckman

50 pH meter. UV-v(V)is spectra were recorded on a

HP8453 spectrophotometer. PO-120 quartz cuvettes

(10mm) were purchased from Shanghai city of China.

Finnpipette Digitals were purchased from Shanghai of

China. BFX5-320 Low Speed Automatic Balance Cen-

trifuge was purchased from Baiyang Centrifuge factory.

Olympus 2700 Complete Automatic Clinical Biochemis-

try Analysis Apparatus was purchased from Japan.

2.3. Measurement Procedure

Using the PV-HEPES-Yb3+ ensemble, we detected inor-

ganic phosphorus in serum and milk samples. The pro-

cedures were as follows. In 10mM, pH 7.0 HEPES

buffer containing 50μM PV and 100μM Yb3+ (a blue

solution), the serum sample from one healthy volunteer

was gradually titrated into the solution. At the same time

the changes in the absorption peaks of solution in the

UV–Vis spectrum were recorded. When no more

changes in the absorption peaks of the system took place,

titration came to a halt. Then we could calculate the in-

organic phosphorus concentration in serum. Likewise,

the inorganic phosphorus concentrations of human milk

degreased or commercial milk were obtained by above

detecting method.

3. RESULTS AND DISCUSSION

3.1. UV–Vis Spectra

Figure 1a shows the UV–v(V)is spectra obtained when

(a)

(b)

Figure 1. (a) The serum from human was added into

PV-HEPES-Yb3+ with Vserum=0-215 μl; (b) the serum from

cow was added into PV-HEPES-Yb3+ with Vserum=0-90 μl.

titrating the serum from a health human into the 10 mM,

pH 7.0 HEPES buffer solution containing 100 μM YbCl3

and 50 μM PV. With the addition of serum, the absorp-

tion peak at 623 nm decreased, while the peak at 444 nm

increased. When the total volume of added serum

reached 210 μL, titration ended. The concentration of

inorganic phosphorus was 0.95 mmol/L. Similarly, Fig-

ure 1b shows UV–v(V)is spectra of serum from cow

titrated. The concentration of inorganic phosphorus from

cow was 2.20 mmol/L. Figure 2a shows UV–v(V)is

spectra changes when titrating milk from healthy woman

into our system. The inorganic phosphorus concentration

of milk from lactation mother was 2.31 mmol/L. Figure

2b shows UV–v(V)is spectra changes of milk from com-

merce process titrated. The inorganic phosphorus con-

centration of milk from commerce was 11.54 mmol/L.

3.2. Selectivity over Other Constituents

In published paper, we addressed the selectivity of the

system. We knew that the ensemble exhibited excellent

C. X. Yin et al. / HEALTH 1 (2009) 76-82

selectivity towards phosphate anions over other common

anions, including Cl−, SO4

2−, CH3COO−, HCO3

− and

ClO4

− [17]. Serum contains many other organic and in-

organic compounds, such as creatinine, bilirubin, sugar,

albumen, inorganic salts and transition ion besides the

aforementioned ordinary anions. Do these compositions

show some responsibility for the changes in the

UV–v(V)is spectra and color? We took the HEPES

buffer as a blank, and then added 200 μL serum into it.

The result shows that there is no UV–v(V)is absorbance

in the range of from 350 to 1,000 nm, suggesting that

there are no other absorbance peaks coming from other

compounds of the serum in the range of detection in the

UV–v(V)is spectra (Figure 3a). Thus, we may conclude

that the changes in the absorbance peak in this range

resulted completely from the measurement processes and

there was no cumulation or disturbance. To prove that

the universal existence of anions in serum incurs no dis-

turbance to exclude the possibility of interference from

iron or other cations in the measurement, the following

experiments were carried out. Firstly, as soon as the ex-

cessive 2 mM YbCl3 was added into the 2 mM HPO4

2−

solution (VYbCl3/VHPO42−=1.02:1), precipitation occurred,

a clear solution was gained through centrifugation and

decantation processes; the solution (from 0 to 500 μL)

was then added into 2 mL 10 mM HEPES buffer con-

taining 50 μM PV and 100 μM Yb3+, and no changes in

absorption peak intensity and color were observed, i.e.,

no phosphate was detected in the solution, thus suggest-

ing that the Yb3+ could completely remove HPO4

2− from

solution by forming sediment. Similarly, we added the

excessive YbCl3 into the collected serum samples whose

content of HPO4

2− was presumably quantitated with our

methods. After the mixture had been treated in accor-

dance with the aforementioned procedures, a great deal

of the disposed serum sample(0–500 μL) was added into

(a) (b)

Figure 2. (a) The milk from woman was added into PV- HEPES-Yb3+ with Vmilk=0-65 μl; (b) the serum from cow was added into

PV-HEPES-Yb3+ with Vmilk=0-13 μl.

(a) (b)

Figure 3. (a) UV/Vis spectra: Plot of absorbance (at λ=200-1000 nm) when adding 20 µl urine into HEPES buffer (pH 7.0); (b) when

adding 50 µl milk into HEPES buffer (pH 7.0).

Openly accessible at

C. X. Yin et al. / HEALTH 1 (2009) 76-82

Openly accessible at

(a) (b)

Figure 4. (a) the disposed serum sample (0-500 μl) was added into PV-HEPES-Yb3+.(b) UV/Vis spectra （λ＝350～800nm）: no

pretreatment serum sample (500 μl) was added into HEPES-PV (50 μM).

the 2 mL 10 mM HEPES buffer containing 50 μM

PVand 100 μM Yb3+, and no changes were observed in

absorption peak intensity and system color(Figure 4a).

The experiments excluded the possibility of interference

from other anions with the measurement. In addition, we

added an adequate amount of unpretreated serum sample

into 2 mL 10 mM HEPES buffer only containing 50 μM

PV, and no change in either the UV–v(V)is spectra or the

system color was observed (Figure 4b). The system was

still yellow. The experiments excluded the possibility of

interference from iron or other cations with the meas-

urement.

Now, we see about the selectivity of the detection

system for milk. We took the HEPES buffer as a blank,

and then added 50 μL milk into it. The result shows that

there is no UV–v(V)is absorbance in the range of from

350 to 1,000 nm, suggesting that there are no other ab-

sorbance peaks coming from other compounds of the

milk in the range of detection in the UV–v(V)is spectra

(Figure 3b). Similary process was done for proving no

disturbance from other composition of milk. All results

ensure that our system is special to phosphate of milk.

3.3. Linearity and Detection Limits

Most instrumental methods available for the determina-

tion of phosphate in clinical samples have a common

drawback; that is, their linear calibration range is too

narrow. In our experiment, we plotted the curve with

absorbance values at 623 nm against concentrations

/0–2.5 mM/ of serum added to the PV-HEPES-Yb3+ sys-

tem. We found our measurement obeyed the Beer–

Lambert absorption law very well within the serum con-

centration range of 0–2.5mM. Linear regression with

least-squares fitting yielded a correlation coefficient of

Figure 5. The working curve for serum measurement was plot-

ted with the absorbance value against various concentrations of

serum(0-2.5 mM).

0.99995 (Figure 5). The lower detection limit of our

method is around 10-4 M. And before, we have gotten

our measurement obeyed the Beer–Lambert absorption

law very well within the urine concentration range of

0–70mM [18]. Phosphorus concentrations of milk from

woman or commerce are not higher than 70mM, so this

linear calibration range is enough for milk.

3.4. Validation

In order to validate the accuracy of the method, we de-

tected serum samples by the standard procedure (mo-

lybdenum blue assay for phosphate) and obtained

equivalent results with our measurement. Figure 6 and

Table 1 give the results (spectra) for serum obtained

with the two kinds of detection methods. Finally, the

recovery experiments were performed: The results are

F. j. Huo et al. / HEALTH 1 (2009) 76-82

Openly accessible at http://www.scirp.org/journal/HEALTH/

(a-1) (a-2)

(b-1) (b-2)

Figure 6. (a)Left: Uv-Vis spectra of cow serum sample Inorganic Phosphorus concentration from our method(2.20 mmol/L); Right:

the results from Molybdenum blue assay for phosphate respectively(2.22 mmol/L; (b) Left: Uv-Vis spectra of serum sample Inor-

ganic Phosphorus concentration from our method (0.95 mmol/L); Right: the results from Molybdenum blue assay for phosphate(0.93

mmol/L).

compiled in Table 2. The results indicated the accuracy

of the method, as expressed by the calculated recovery

values, was satisfactory.

3.5. Analysis of Results

From our data, we can see inorganic phosphorus content

of milk is higher than foremilk about five times. This

indicates the excessive inorganic phosphorus is disad-

vantageous to young child’s growth. And with the people

level of living enhancement, the commercial milk be-

comes the people basic nutriment. But in the processing

commercial milk many important ingredients content are

insufficient, for instance, the calcium, phosphorus ratio

of is 2: 1 in milk containing the few calcium, many

phosphorus, is easy to form the insoluble calcium phos-

phate, affects the intestinaltract absorbing calcium and

phosphorus. If provide turnips containing many calcium,

few phosphorus for the child who eats the milk, can cor-

rect calcium and phosphorus proportion, namely can

enhance the calcium absorbing capacity. Therefore, the

reasonable increase and the adjustment can only prevent

to be out of nutrition balance for one people drink milk.

3.6. Assay Advantage

Ion-based chromogenic method has proved to be a useful

tool for clinical analysis because of its simplicity, re-

peatability, low reagent consumption and so on. We

know that ion chromatography, the use of deproteinizing

agents presents several disadvantages: perchloric acid

and trichloroacetic acid need to be removed from the

sample by time-consuming procedures otherwise they

C. X. Yin et al. / HEALTH 1 (2009) 76-82

Openly accessible at

Table 1. Results of our method against the method (molybde-

num blue assay for phosphate).

Numbers of

serum sample

Phosphate

(mmolL-1) from

Molybdenum blue

assay

Phosphate

(mmolL-1) from

our method

1(Serumhuman) 0.96 0.97

2(Serumhuman) 0.93 0.95

2(Serumcow) 2.20 2.22

Table 2. Recovery of phosphate in serum and milk.

interfere with the elution profile; organic solvents such

as acetonitrile lead to damage to the column and sul-

phosalicylic acid is often contaminated with sulphate,

lead to sample dilution. And our method decreases the

cost of analyses with respect to batch methods involving

enzymes in solution. All these advantages make the

method reported here be a valid alternative for the de-

termination of phosphate in serum.

4. CONCLUSIONS

To sum up, we developed a sensitive, rapid and direct

method for detecting serum and milk phosphate spec-

trophotometrically. Our method is suitable for perform-

ing direct determinations of phosphate in serum without

any pretreatment and any interference. Now more and

more people are suffering from lithiasis as a result of

better living standards. Timely inspection of serum

phosphate is one of the clinical means of diagnosis.

Since long-time, people only pay attention to the inor-

ganic phosphorus determination in the urine and the

blood serum, to determine the inorganic phosphorus in

the foremilk mother’s milk and milk method very little

was mentioned. In this paper, we use our invention sys-

tem to quantificationally determine inorganic phospho-

rus concentration from milk degreased through centrifu-

gation, the results are accurate, suit to clinical and the

commerce use.

5. ACKNOWLEDGEMENTS

We acknowledge to the financial support of this work by the

National Natural Science Foundation of China (No. 20801032),

Shanxi Provincial Natural Science Foundation (No.

2009021006-2) and the Shanxi Province Foundation for Re-

turness (2008).

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Sample

Added

(phosphate)

mmolL-1

Found

(phosphate)

mmolL-1

Recovery

(%)

1(Serum) — 1.48

0.6 2.02 95.9

2(Serum) — 1.11

0.6 1.73 102

3(Serum) — 0.95

0.6 1.53 97.3

4(Milkhuman) — 2.34

1.0 3.42 104

5(Milkcow) — 12.5

5 17.3 98.4

C. X. Yin et al. / HEALTH 1 (2009) 76-82

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