Determination of Inorganic Phosphorus in Human Blood Serum
by use  Square Wave Voltammetry (SWV)                

Amer Th. Al-Taee and Ramiz S. Kassir
Department of Chemistry, College of Science, University of Mosul
 Mosul , IRAQ

(NJC)

(Received on  25/11 /2004)		(Accepted for publication on 22/ 6/2005)

Abstract
	A square wave voltammetric method is described for the determination of Inorganic Phosphorus in human blood serum. This method is based on the decrease on the reduction peak of ferrous ammonium sulphate Fe(NH4)2(SO4)2.6H2O appeared at (-0.2 V) vs. (Ag/AgCl,3M KCl) as a reference electrode using (Sodium acetate, acetic acid) buffer adjusted by (1.0 N) NaOH to (pH 7.0) as supporting electrolyte due to its consumption via the enzymatic reaction of Inorganic Phosphorus. The method was successfully applied to measure the Inorganic Phosphorus in various blood samples represent different cases such as: hyperparathyroidism, rickets (in children), osteomalacia (in adults), chronic renal failure, Addison’s disease and diabetic ketoacidosis.
	The results have been compared with those obtained from the spectrophotometric method and show a good agreement between the two methods with correlation coefficient (r = 0.9996). 
الخلاصة
		تتضمن الطريقة تقدير كمية الفوسفات اللاعضوية  (Inorganic Phosphorus) في مصل الدم البشري باستخدام طريقة فولتامتري الموجة المربعة (SWV). حيث تعتمد على قياس الانخفاض في القمة البولاروغرافية لاختزال مادة كبريتات الامونيوم الحديدوزي (ferrous ammonium sulphate) المستهلك خلال التفاعل الانزيمي للفوسفات اللاعضوية والتي تظهر عند جهد (-0.2) فولت ضد قطب المرجع (فضة / كلوريد الفضة، 3 مولاري كلوريد البوتاسيوم) باستخدام محلول (خلات الصوديوم، حامض الخليك) المنظم مع بعض القطرات من (1 عياري) هيدروكسيد الصوديوم للوصول الى الرقم الهيدروجيني (pH=7.0) كعامل مساعد . حيث تم تطبيق الطريقة بنجاح لقياس كمية الفوسفات اللاعضوية في عينات مصل الدم لاشخاص اصحاء وكذلك المصابين في حالات مرضية مختلفة مثل : زيادة افراز الغدة الدرقية، مرض الكساح (لدى الاطفال)، مرض نخر العظام (لدى الكبار)، التهاب الكلية المزمن، مرض اديسون و مرض السكري وكذلك تم مقارنة النتائج المستحصلة مع الطريقة اللونية وكانت العلاقة خطية بمعامل ارتباط (r = 0.9996).

Introduction
Phosphorus is a group 5 element of the periodic table and has an atomic weight  of 30.97. It is most commonly found in nature in its pentavalent form in combination with oxygen, as phosphate (PO43-). Phosphorus is widely found in many food groups, largely as phosphate(s). Dietary sources that are rich in phosphorus include red meats (1600 mg/kg), dairy products            (> 900 mg/kg), fish (4000 mg/kg), poultry (2100 mg/kg) and bread and other cereal products  (> 900 mg/kg). A number of phosphate salts are used in foods and soft drinks as additives (1). Phosphorus is also used in food supplements (at levels up to a daily dose of 1100 mg/day) and licensed medicines, in the form of inorganic phosphate salts and sodium acid phosphate, respectively. (2)
Phosphorus is abundant in the body with largest amounts found in bone. It is also found in all soft tissues including muscle, liver, heart and kidneys. Approximately 80% of the body phosphorus is present in the skeleton and the remainder is distributed in soft tissues and extracellular fluid. About 70% of the phosphorus in blood is as a constituent of phospholipids; the remainder is present as inorganic phosphate (HPO4-2), about 85% free and 15% protein-bound (3,4). It is a constituent of all major classes of biochemical compounds. Structurally, phosphorus occurs as phospholipids, which are a major constituent of most biological membranes, and as nucleotides and nucleic acids. Phosphorus plays an important role in carbohydrate, fat and protein metabolism and is essential for optimum bone health.(5,6)
The Normal levels of serum phosphorus range 2.5-4.5 mg/dl (0.81-1.45 mmol/l). Increased levels of serum phosphorus are seen in renal disease, hypoparathyroidism and excessive Vitamin D intake. Decreased levels are seen in rickets, osteomalacia (adult rickets), hyperparathyroidism and diabetic coma (7-10), Lastly, Phosphorus is primarily excreted in the urine. The regulation of phosphorus excretion is apparent from early infancy. In infants, as in adults, the major site for the regulation of the amount of phosphorus retained by the body is the kidney. (11)
	Square Wave Voltammetry (SWV) is a pulsed voltammetry technique that uses a potential waveform as shown in [Fig.(1)]. The advantage of square wave voltammetry is that the entire scan can be performed on a single mercury drop in about 10 seconds, as opposed to about 5 minutes for the techniques described previously. SWV saves time, reduces the amount of mercury used per scan by a factor of 100. If used with a pre-reduction step, detection limits of 1-10 ppb can be achieved.

           

Fig.(1): The Relationship between Potential  & Time for Square Wave Voltammetry (SWV) (12)

The data for SWV appear much the same as data for Differential Pulse Polarography (DPP), although the height and width of the wave depends on the exact combination of experimental parameters (i.e. scan rate and pulse height), unlike DPP. This feature makes the running of standards important, as with any analytical technique. Like DPP, the current at the beginning of a pulse is subtracted from the current at the end of a pulse. (13-16)

	Many methods are available for determination of Inorganic Phosphorus in human blood serum(17,18) . The colorimetric method (UV Phosphomolybdate method) is based on the formation of  as shown (19):

	This method required different reagents for color development and its sensitive too much lower than electrometric method.

The Differential Pulse Anodic Stripping Voltammetry (DPASV) had been applied to measure the activity of  Lactate Dehydrogenase (LDH) enzyme in human blood serum. (20)
	
In the present work, SWV is used for determination of Inorganic Phosphorus quantity in human blood serum as described in the following procedure.
   
Experimental
Reagents
1.	 Reagent 1 (Standard) from P-Kit (Ref. 61 599) (bioMerieux) containing :
Phosphorus (1.61 mmol/l) (5 mg/100ml – 50 mg/l) and Sodium azide (1 g/l) in a total volume (5 ml).

2.	 Reagent 2 (Reducting agent ) from P-Kit (Ref. 61 599) (bioMerieux) containing :
Sulfuric acid (1.06 N), ferrous ammonium sulphate (100 g/l) and ferrous nitrate (2g/l) in a total volume (50 ml).

Reagent 3(Color Reagent)  from Ref. (61 599) (bioMerieux) containing: 
Sulfuric acid (1.05 N) and ammonium hepta-molybdate (4.5 g/l) in a total volume (50 ml).

3.	(Sodium acetate, acetic acid) Buffer (0.2 M) at (pH 7.0).
Freshly prepared by mixing (9.5 ml) of CH3COONa and (0.5 ml) of CH3COOH diluted to (100 ml) by Distill water (pH = 5.9), Then the pH adjusted to (7.0) by adding few drops of (1.0 N) NaOH solution.(21)

4.	Sodium Hydroxide NaOH (4.0 N) from (Cat. No. AS 147,)(Randox) containing :
    Sodium Hydroxide (4.0 N) in a total volume (100 ml).

5.	Ferrous Ammonium Sulphate (10-3 M) from (Fluka): 
     Freshly prepared by dissolving (0.0098 gm) of  Fe(NH4)2(SO4)2.6H2O in (25 ml) of distilled water in a volumetric flask.

6.	(Precision  Multi-Sera  Low Human)(Cat .No. UL2701)(Randox):
	Reconstitute each vial of lypophilised serum with exactly (5 ml) of distilled water. then stand for (30 min.) out of bright light before use.

7.	(Precision  Multi-Sera Normal Human)(Cat. No. UN 1557) (Randox):
	Reconstitute each vial of lypophilised serum with exactly (5 ml) of distilled water. then stand for (30 min.) out of bright light before use.

8.	(Precision  Multi-Sera Elevated Human)(Cat. No. UE 1558) (Randox):
	Reconstitute each vial of lypophilised serum with exactly (5 ml) of distilled water. then stand for (30 min.) out of bright light before use.

Specimen Collection and Preparation
	Samples of human serum were obtained from routine clinical assays. Serum samples were prepared and assayed within (1 hr), otherwise the serum should be kept frozen.

Apparatus
		All the voltammetric measurements were performed using (Computerized Polarographic Analyzer) from (EG&G) company which include (HMDE) model (303A) and (Digital Plotter) model (DMP 04-44). The voltammetric cell consists three electrodes: the working electrode was a Hanging Mercury Dropping Electrode (HMDE), the reference electrode was (Ag/AgCl,3M KCl) and a Pt wire  as a auxiliary electrode. Temperature control made by using radiometer VTS 13 water thermostat (±0.1 0C).
		But the colorimetric measurements were performed by using  (Cecil Spectrophotometer) model (CE 10211 Ultra Violet & Visible Spectrohotometer) from Cecil Instruments Limited. 

Procedure
		The optimum conditions were examined using (SWV) were show in Tables (1-4) as: Deposition time (5 s), Conditioning time (0 s), Equilibrium time (5 s), Scan Rate (100 mV/s). The voltammetric cell was thermostated at (37oC). The solution was deaerted by passing a slow stream of pure N2 gas through it for (4 min.) to remove the dissolved oxygen.
		 For voltammetric measurement, the sample cuvette contained (5ml) of (0.2 M) (Sodium acetate, acetic acid) Buffer (pH 7.0) and (100µl) Reagent 2. The square wave voltammogram of ferrous ammonium sulphate was recorded between (0.0 V to -0.4 V).and the reaction was initiated by addition of (5µl) of serum. The voltammogram was again recorded. The calculation of voltammetric method (SWV) were show in the flowing equation:-

The concentration of Inorganic Phosphorus  = x n , which in :-

Ip: is the value of diffusion current of ferrous ammonium sulphate before serum addition, the unit of Ip is nanoAmper (nA).

Ip1: is the value of diffusion current of ferrous ammonium sulphate after serum addition, the unit of Ip1 is (nA).

Ip standard: is the value of diffusion current of the standard, the experimental value of  Ip standard equal to 1000 nA.

 N: is the value of the standard concentration equal to 1.61 mmol/l.

	For colorimetric determinations, we prepared working solution by mixing 1 volume of Reagent 2 with 1 volume of Reagent 3, the stability of working solution is one month at            2-8 oC in a dark bottle. The wavelength that used in colorimetric method was 690 nm (Hg 691). We used distilled water as reagent blank to made zero adjustment. The procedure that used in colorimetric method could be shown in the following list :-

	Reagent blank	Standard	Sample	
Sample	----------	----------	100 µl	
Reagent 1 (standard)	----------	100 µl	----------	
Distilled water	100 µl	----------	----------	
Working solution	2.5 ml	2.5 ml	2.5 ml	

	After prepared three tubes for each sample as shown in the above, we were mixing these tubes and waiting for 10 minutes then we measured the absorbance for each sample. The color intensity was stable about one hour. The calculation of colorimetric method were show in the flowing equation:-

The concentration of Inorganic Phosphorus = x n , which in :-

A Sample: is the value of the sample absorbance.

A Standard:  is the value of the standard absorbance, the experimental value of  A standard equal to 0.195

N: is the value of the standard concentration eq ual to 1.61 mmol/l.

Results and Discussion
		Colorimetric determination, without deproteinization of serum phosphrous using a single reagent which forms a phosphomolybdate complex in the presence of a reducing agent (ferrous sulphate).(19)
		In voltammetric method, the principle of SWV method is based on the reduction process of ferrous ammonium sulphate on the surface of HMDE when combining with inorganic phosphorus in serum to form the complex between them as shown in the following equation:-

Ferrous ammonium sulphate

Complex

Inorganic Phosphorus

Fe(NH4)2(SO4)2.6H2O +   HPO4-2                   Fe(NH4)2(HPO4).6H2O + SO2 + ½ O2 - 2e-

Table (1): Effect of Deposition Time (Dep. Time) on the reduction peak of ferrous   ammonium sulphate at Ep = -0.2 V

Dep. Time (sec.)	0	5	10	15	20	
Ip (nA)	6275	8120	7261	5439	5274	

Table (2): Effect of Conditioning Time (Cond. Time) on the reduction peak of ferrous ammonium sulphate at Ep = -0.2 V

Cond. Time (sec.)	0	5	10	15	
Ip (nA)	8425	7375	7329	6045	

Table (3): Effect of Equilibrium Time (Equi. Time) on the reduction peak of ferrous ammonium sulphate at Ep = -0.2 V

Equi. Time (sec.)	5	10	15	20	
Ip (nA)	8546	8035	7817	7514	

Table (4): Effect of Scan Rate on the reduction peak of ferrous ammonium sulphate          at Ep = -0.2 V

Scan Rate (mV/sec)	100	200	300	400	
Ip (nA)	8945	7920	6811	5787	

		From the above equation, Fe(II) is reduce to Fe(0) on the surface of  HMDE then forming the complex. Therefore after addition of (100µl) Reagent 2  to the cuvatte sample which contain (5 ml) of (sodium acetate, acetic acid) Buffer at (pH 7.0), we found that ferrous ammonium sulphate gives a well known defined peak at (-0.2 V) vs. Ag/AgCl, 3M KCl in (Sodium acetate, acetic acid) Buffer at (pH 7.0) [Fig. 2(b)] and can be used for indirect determination of Inorganic Phosphorus by measuring the decrease in this peak after addition of (5 µl) of serum as shown in [Fig. 2(c)].
 
Optimization of Conditions:

Effect of  (10-3M) Volume of  Ferrous Ammonium Sulphate Solution:	
		In order to determine the reduction peak of ferrous ammonium sulphate that           appeared at the reduction potential (-0.2 V), a series of experiments were carried out in which (10-3M) Ferrous Ammonium Sulphate solution added in a range between (10-100µl) to a voltammographic cell containing (5 ml) of (sodium acetate, acetic acid) Buffer at (pH 7.0). The results shows increasing in the diffusion current (Ip) of the FeSO4(NH4)2SO4.6H2O at (-0.2 V). therefore the result for this experiment proved that the reduction peak is related to ferrous ammonium sulphate. (Table 5)

Table (5): Effect of (10-3M) volume of Ferrous Ammonium Sulphate solution on the reduction peak of ferrous ammonium sulphate at Ep = -0.2 V
Addition of (10-3M) Ferrous- Ammonium Sulphate solution (µl)	                             Ip (nA)	
10	3650	
20	3970	
30	4217	
40	4495	
50	5360	
60	5718	
70	6030	
80	6235	
90	6785	
100	7120	

a

Peak current (Ip, nA)

Potential (Ep, V)

Potential (Ep, V)

b

c

Peak current (Ip, nA)

Peak current (Ip, nA)

Potential (Ep, V)

b

a

Fig.(2): Square-wave Voltammograms :-
              
(a) For the supporting electrolyte only (Sodium acetate, acetic acid) buffer at pH=7.0 
(b) For the Ferrous Ammonium Sulphate before the addition of human blood serum.
(c) For the Ferrous Ammonium Sulphate after the addition of human blood serum,(shows the decrease in the reduction peak).

Effect of Reagent 2 (Reducting agent) 
		In order to determine the optimum concentration of ferrous ammonium sulphate, a series of experiments was carried out in which Reagent 2 solution added in a range between (10-100µl) to a voltammographic cell containing (5 ml) of (Sodium acetate, acetic acid) Buffer at (pH 7.0). The result shows that (100µl) of Reagent 2 solution is suitable to determine the inorganic phosphorus quantity in clinical application due to the maximum diffusion current (Ip). (Table 6)

Effect of Reagent 1 (Standard)
		The effect of Reagent 1 examined by adding increasing amount of standard in a range between (1-5µl) to a voltammographic cell containing (5 ml) of (Sodium acetate, acetic acid) Buffer at (pH 7.0), and (100µl) of Reagent 2 solution. The result shows that (5µl) of standard represent the optimum amount for determination because giving the maximum value of diffusion current (Ip). (Table 7)

Effect of Serum 
		The effect of Serum examined by increasing amount of normal human serum in a range between (1-5µl) to a voltammographic cell containing (5 ml) of (Sodium acetate, acetic acid) Buffer at (pH 7.0), and (100µl) of Reagent 2 solution. The result shows that (5µl) of serum represent the optimum amount for determination because it gives the maximum value of diffusion current (Ip). (Table 8)

Clinical Applications 
		A comparison between SWV method and spectrophotometric method was carried out using (36 Samples) represent a normal cases and patients suffered from different  diseases such as: hyperparathyroidism, rickets (in children), osteomalacia (in adults), chronic renal failure, Addison’s disease and diabetic ketoacidosis. The results obtained are shown in Table (11) .
		The plot of concentration  measured by SWV method versus that measured by colorimetric method that seen in [Fig. (3)] gives a straight line with correlation coefficient (r = 0.9996) and (RSQ = 0.9992) that’s indicates the good agreement between the two methods and the relation between them can be represented by the following equation:

Conc. by SWV method = 0.0084 + [1.2209 * Conc. by Colorimetric  method]
Quality Control
		For accuracy and reproducibility control, we assayed Multi-Sera Low, Normal and Elevated by the two methods: SWV and Colorimetric methods. The results shown in (Table 9).
		For ensure the accuracy of our purposed voltammetric method (SWV method), we calculated each of : Related Standard Deviation (RSD%), Relative Error (Error%) and (Recovery%) to increase the accuracy of our purposed method, the results shown in (Table 10).      

Table (6): Effect of Reagent 2 amount on the reduction peak of ferrous ammonium sulphate at Ep = -0.2 V
Addition of Reagent 2 (µl)	                             Ip (nA)	
10	6990	
20	7030	
30	7114	
40	7800	
50	7970	
60	8250	
70	8545	
80	8820	
90	9180	
100	9390	

Table (7): Effect of Reagent 1 on the reduction peak of ferrous ammonium sulphate  at Ep = -0.2 V

Addition of Reagent 1  (µl)	1	2	3	4	5	
Ip (nA)	9000	8360	7980	6990	6260	

Table (8): Effect of Serum on the reduction peak of ferrous ammonium sulphate  at Ep = -0.2 V

Addition of Serum  (µl)	1	2	3	4	5	
Ip (nA)	8975	8250	7815	7120	6760	

Table (9): A Quality Control of Inorganic phosphorus quantity by the two methods:  [(SWV method) and (Colorimetric method)].

Quality Control Standards	Range (mmol/l)	Colorimetric Method (mmol/l)	SWV Method (mmol/l)	
Low Human Sera	0.50-0.74	0.687	0.850	
Normal Human Sera	1.20-1.80	1.510	1.869	
Elevated Human Sera	1.75-2.60	2.502	3.096	

Table (10): The statistical results for accuracy of purposed voltammetric method (SWV method).
 
	RSD %	Error %	Recovery %	
Low Human Sera	0.153416	0.94118	99.05882	
Normal Human Sera	0.091658	0.32103	99.67897	
Elevated Human Sera	0.041787	0.22610	99.77390	

Table (11): The results obtained for determination of Inorganic Phosphorus in Normal and Abnormal cases using two methods: SWV method and Colorimetric method.

Voltammetric Method (SWV method)	Colorimetric Method	
Sample No.	Ip (cm)	Ip1 (cm)	Ip-Ip1 (cm)	Conc. (mmole/l)	A Sample	Conc. (mmole/l)	
1	8370	7209	1161	1.869	0.183	1.510	
2	8370	7982	388	0.625	0.065	0.538	
3	8370	7909	461	0.742	0.076	0.624	
4	8369	7881	488	0.785	0.078	0.648	
5	8370	7827	543	0.875	0.083	0.687	
6	8368	7809	559	0.900	0.090	0.741	
7	8371	7768	603	0.970	0.095	0.784	
8	8370	7735	635	1.022	0.100	0.826	
9	8372	7731	641	1.032	0.102	0.842	
10	8371	7654	717	1.155	0.113	0.933	
11	8370	7618	752	1.210	0.117	0.966	
12	8371	7597	774	1.247	0.122	1.007	
13	8369	7537	832	1.339	0.131	1.082	
14	8370	7507	863	1.390	0.136	1.123	
15	8371	7476	895	1.441	0.141	1.164	
16	8370	7431	939	1.512	0.148	1.222	
17	8368	7297	1071	1.725	0.170	1.404	
18	8369	7252	1117	1.799	0.176	1.453	
19	8370	7223	1147	1.847	0.181	1.492	
20	8368	8098	270	0.434	0.043	0.351	
21	8370	7209	1161	1.869	0.192	1.585	
22	8370	7145	1225	1.972	0.193	1.593	
23	8369	7036	1333	2.146	0.210	1.734	
24	8366	6841	1525	2.456	0.241	1.990	
25	8368	6807	1561	2.514	0.246	2.031	
26	8370	6754	1616	2.602	0.255	2.102	
27	8371	6740	1631	2.626	0.257	2.122	
28	8372	6497	1875	3.018	0.303	2.502	
29	8370	6171	2199	3.540	0.366	3.022	
30	8369	5671	2698	4.343	0.425	3.509	
31	8369	5531	2838	4.569	0.447	3.694	
32	8368	5179	3189	5.135	0.524	4.327	
33	8369	4959	3410	5.490	0.548	4.523	
34	8370	4822	3548	5.712	0.560	4.624	
35	8371	4808	3563	5.736	0.564	4.658	
36	8370	4379	3991	6.425	0.630	5.202	
Which in:-
Conc. is the Concentration of  Inorganic Phosphorus in human blood serum measured by two methods: SWV method and Colorimetric method. 

Fig.(3) : The relation between Concentrations of Inorganic Phosphorus measured by SWV method and Colorimetric method in Normal & Abnormal cases.

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