Quantitative determination of ethane-1-hydroxy-1,1-diphosphonate in urine and plasma

299

Clinica Chimica Acta, 65 (1975) 299-307 0 Elsevier Scientific Publishing Company,

Amsterdam

-Printed

in The Netherlands

CCA 7366

QUANTITATIVE DETERMINATION OF ETHANE-l-HYDROXY-l,l-DIPHOSPHONATE

SYLVIA

BISAZ, ROLF FELIX and HERBERT

Department of Pathophysiology, (Switzerland) (Received

IN URINE AND PLASMA

FLEISCH

University of Berne, Murtenstrasse 35, 3008 Bern

June 12,1975)

Summary A technique has been developed to measure the diphosphonate ethane-lhydroxy-l,l-diphosphonate (EHDP) quantitatively in 5 ml of urine or 2 ml of plasma. The procedure is based on a coprecipitation of EHDP with calcium phosphate, elimination of inorganic phosphate as an insoluble triethylaminephosphomolybdate complex, decomposition of the P-C-P bond with ultraviolet light and spectrophotometric determination of the inorganic phosphate released. A trace amount of [“Cl EHDP is used to correct for losses. The method appears specific for the diphosphonate, exhibits quantitative recoveries, and has a mean coefficient of variation of 3.7% for urine and 7.3% for plasma. The limit of detection is in the order of 2.5 pmol/l in 5 ml urine and 0.5 pmol/l in 2 ml of plasma.

Introduction Recently a new class of compounds, the diphosphonates, have been introduced in the treatment of diseases involving calcium metabolism. These compounds have been found to inhibit ectopic calcification of soft tissues [l] and to slow down bone resorption in experimental animals [2]. In man, favorable effects have been found in myositis ossificans progressiva [3] and in Paget’s disease [4-61. Laboratory and clinical studies with diphosphonates have become increasingly hampered by the lack of a simple chemical technique for measuring diphosphonates, particularly ethane-l-hydroxy-1,1-diphosphonate (EHDP) which is the only one so far used in man. The only chemical technique described until now [ 71 is not sensitive enough for plasma and requires a volume of 500 ml of urine for one determination. This paper describes a new technique for measuring micromolar concentrations of EHDP in 5 ml of urine or 2 ml of plasma. It is based on the copreci-

300

pitation of EHDP with calcium phosphate, elimination of inorganic phosphate as insoluble triethylamine-phosphomolybdate complex, decomposition of the P-C-P bond by ultraviolet photolysis, and spectrophotometric measurement of the inorganic phosphate formed. Materials Chemicals were obtained from Merck Chemical Company, Darmstadt, WGermany. The EHDP as well as the [‘“Cl EHDP (spec. act. 1.97 Ci/mol) were a gift from the Procter and Gamble Company, Cincinnati, Ohio, U.S.A. The following equipment was used: Packard 3375 sc~tillation counter. UV-lamp Hanau Q400, a quartz high pressure (5 atm) mercury-vapor lamp, 120 W. The irradiation is performed in quartz tubes (internal diameter 12-14 mm) placed at 10 cm from the UV-source. Filtration: Sartorius Memb~nfilters 11303 (G~ttingen) were used for urine, and Millipore-Swinnex-13 devices with HAWP 01300 filters for plasma. Spectrophotometers: for urine, a Gilford spectrophotometer adapted on Beckman DU monochromator and Gilford 2443-A rapid sampler; for plasma, Zeiss spectrophotometer PMQ II with microcells 5 cm in length. containing polyphosphates have to be strictly Glassware : detergents avoided when washing the glassware. Methods The different

steps of the method

are given in Fig. 1.

Urine The urine is first adjusted to pH 3 by dropwise addition of concentrated HCl in order to dissolve any calcium phosphate or ammonium magnesium phosphate which could adsorb EHDP. It is then filtered through a paper filter (Schleicher and Schiill 595 was used but the type is probably not critical). 5 ml of the urine are transferred to a centrifuge tube and 100 ~1 of 30 pmol/l [14C J EHDP, corresponding to about 6 nCi 14C, added in order to correct for subsequent losses of EHDP. The two precipitation steps which follow (Step I) allow the EHDP to be separated from many of the salts and other substances contained in the urine and are based on the observation that calcium phosphate binds EHDP very strongly [8]. After addition of 50 ,ul of 2.5 mol/l CaC&, 1 mol/l NaOH is added dropwise while shaking until precipitation starts; a small precipitate is sufficient to coprecipitate EHDP. The solution is centrifuged 10 min at 3000 X g, the supernatant discarded and the precipitate dissolved in 2 ml 2 mol/l HCl. After addition of 8 ml of water, calcium phosphate is once more precipitated by dropwise addition of 10 mol/l NaOH until a precipitate appears. After centrifugation, the sediment is redissolved in 2 ml of 2 mol/l HCl and heated in a water bath for 30 min at 100°C (Step II) in order to hydrolyse pyrophosphate and other acid-labile phospha~ compounds into inorganic phosphate (Pi). Pi is then removed from the solution through formation of an insoluble triethylamine-phosphomolybdate complex [ 9 ] (Step III). For this

301

Urine + [~~C]EHDP

1

Precipitation of EHDP with (repeated 1

Dissolution of tha precipitate

step I

with

step II

3Dmin at lDD*C Precipitation

of Pi with

NH,-molybdate + triethylamine chloride

Precipitation of EHDP with

Dissofution af the pncipitatt

step m

step H

with

Q

Urine: lDml of extract Plasma: 2.2ml af extract

SttpY

‘.C determination

2 h W-photolytis

t

1

f+ determination

stepllx 1

step Qn

Fig. 1. Scheme of the method used.

step it is necessary to know how much Pi is present, so that the correct amounts of ~monium molybdate and triethyl~ine chloride are added. For this 8 ml of water are added and the Pi -content of the solution measured in an aliquot of 20 ~1, by the technique of Bisaz et al. [lo]. The calculation of the necessary amounts of reagents is based on the fact that the theoretical quantities necessary to precipitate 20 ,umol Pi are 1 ml 40 mmolfl ammonium molybdate and 0.1 ml 0.8 mol/l triethylamine chloride, the precipitate consisting of 1 mol Pi, 2 mol molybdate and 3 mol triethylamine [ 9 J . 100 ~1 of bromine water (0.5 ml bromine dissolved in 10 ml of water) are then added (in order to oxidize any blue molybdate complexes formed), folfowed by 15-20% more than the calculated amount of the two Pi precipitating reagents. The volume of molybdate needed is generally between 1.5 and 2.5 ml. It is better not to produce too large a precipitate of calcium phosphate, i.e. not to add too much NaOH in the first two precipitation steps, because the triethylamine-phosphomolybdate complex is very voluminous and some of the solution is trapped in the precipitate and therefore lost to the analysis at this

302

stage. After standing 5 min in ice-water, the mixture is centrifuged 10 min at 3000 X g and the supernatant filtered through a Sartorius filter 11303 to make sure that all the precipitate is removed. The filtrate is placed in a centrifuge tube and 100 ~1 bromine water and 100 ~12.5 mol/l CaCl, are added, followed again by dropwise addition of 10 mol/l NaOH just until a light precipitate is formed. This precipitate consists of Ca(OH), ; EHDP is also coprecipitated (Step IV). The solution is centrifuged, the supernatant discarded and the precipitate dissolved in 200 ~1 1 mol/l HCl and diluted with water to give 10 ml of extract (Step V). The radioactivity of 14C in this extract is measured in a 100 ~1 aliquot added to 10 ml of scintillator (naphthalene 80 g; toluene 600 ml; methylcellosolve 400 ml; butyl-PBD (2-(4-tert-butyl-phenyl)-5-(4_biphenylyl)-1,3,4oxadiazol) 7 g). The comparison of this activity with the activity initially added permits the calculation of the EHDP recovery. EHDP is then decomposed to Pi by UV-photolysis [ 111 for 2 hours (Step VI) on the following samples: (a) 1 ml of extract + 1 ml of water; (b) 2 ml of extract; (c) 1 ml of extract + 1 ml of 20 pmol/l unlabelled EHDP, in order to check the efficiency of the photolysis. The samples (a) and (b) are prepared in duplicate: one is subjected to photolysis, the other is kept in the dark and used as a blank to measure the trace amounts of Pi not precipitated in the complex. After irradiation Pi is determined in all the samples spectrophotometrically (Step VII): 80 ~1 of 12 mol/l HCl and 2 ml of molybdate reagent (4 mmol/l ammonium molybdate and 114 mmol/l ascorbic acid in 1 mol/l HCI are added, the tubes are heated for 10 min at 100°C in a water bath and after cooling, the absorbance is measured at 820 nm [lo]. The difference in the Pi contents of the irradiated sample and blank corresponds to the amount of EHDP. A mean of the value obtained with the 1 ml and 2 ml samples is taken and corrected for recovery and for added [‘*Cl EHDP. Plasma

To 2 ml of plasma are added 100 ~112 pmol/l [ 14C] EHDP, corresponding to about 2.4 nCi ’ 4C. Proteins are precipitated with 2.1 ml 0.61 mol/l trichloroacetic acid and centrifuged for 20 min at 48 000 X g in a Sorvall centrifuge RC 2-B. 10 mol/l NaOH is then added dropwise to the supernatant until a small precipitate appears (Step I). After centrifugation for 10 min at 3000 X g the precipitate is dissolved in 2 ml 1 mol/l HCl, 2 ml of water are added and a second precipitation induced by 10 mol/l NaOH. After centrifugation and redissolution of the precipitate with 1.0 ml 1 mol/l HCl, hydrolysis is performed as for urine (Step II). 0.5 ml 1 mol/l NaOH and 1.5 ml Hz0 are then added to produce optimal conditions for a nearly quantitative precipitation of Pi, which decreases the absorbance of the blank. This is more critical than for urine because of the small volume of the final extract (2.2 ml instead of 10 ml) and of the determination of Pi which is 5 times more sensitive in the 5 cm length cells. The best concentration of HCl is between 0.1 and 0.2 mol/l. Since the phosphate content in plasma is less variable than in urine, no previous determination of Pi

303

is necessary. With 2 ml of normal human plasma, the solution contains at this stage about 1 pmol Pi which theoretically needs 50 ~1 of 40 mmol/l molybdate for precipitation. This Pi is precipitated with 100 ~1 of 40 mmol/l ammonium molybdate and 10 ~1 of 0.8 mol/l triethylamine chloride (Step III). If the Pi content of the plasma is increased to 1.5-2 mmol/l (in patients with renal failure or on chronic EHDP intake, or in rat plasma), the amount of the precipitating reagents has to be doubled. After this Pi precipitation step, the glassware used has to be phosphatefree and should thus have been washed carefully with 0.5 mol/l HCl. The solution is left in ice-water for 5 min, the precipitate is discarded after centrifugation. After standing 5 min the supernatant is filtered through a Millipore-Swinnex-13 device using a filter HAWP 01300. 50 111of bromine water are added to the filtrate, followed by 50 ~1 2.5 mol/l CaCl, and 4 mol/l NaOH dropwise until precipitation occurs (Step IV). After a short centrifugation the supematant is discarded and the precipitate immediately dissolved in 50 ~1 of 1 mol/l HCl, 50 ~1 of bromine water and 2.1 ml Hz0 to give a final extract of 2.2 ml (Step V). 100 ~1 are taken for determination of radioactivity. 1 ml is irradiated for 2 hours as for urine and 1 ml kept in the dark (Step VI). To both samples 20 ~1 of 12 mol/l HCl and 1.0 ml of the molybdate reagent are added. After heating the solutions for 10 min at lOO”C, the absorbance is measured in a Zeiss spectrophotometer at 820 nm using microcells with a 5 cm lightpath (Step VII). Plasma EHDP content is calculated in a similar way as for urine, taking into account the recovery and the amount of [‘“Cl EHDP added. Results and discussion

Recovery of EHDP during the isolation procedure The recovery of [ 14C] EHDP through the procedure lies between 70 and 90% for urine and between 60 and 80% for plasma. This variation emphasizes the necessity of using an isotope dilution method with radioactive EHDP so that corrections for losses are easy to make. Efficiency Of Pi precipitation The method of Sugino and Miyoshi [9] used to precipitate Pi proved to be very efficient: thus solutions with a concentration of 1000,100,10 or 1 pmol/l Pi contain after precipitation 21,2, 0.4 or 0.1 pmol/l Pi, respectively. This nearly quantitative precipitation of phosphate is necessary, in order to reduce the phosphate content of the solution before photolysis. Since the estimation of EHDP involves the difference between untreated samples and those subjected to photolysis, it would be difficult to measure low concentrations of EHDP with precision if large amounts of Pi are left over from previous steps. Photolysis by UV light Efficiency. To check the efficiency of the photolysis 32 samples with EHDP concentrations varying from 0.4 to 40 pmol/l were irradiated for 2 hours and analysed as Pi. For low concentrations (between 0.4 and 4 pmol/l) measured in the microcells on the Zeiss spectrophotometer, the mean of the recov-

304

ery of added EHDP was 99.35% for 12 determinations (SD. = 4.85). The correlation coefficient between theoretical and determined concentration was r = 0.9990. For higher concentrations (between 4 and 40 pmol/l) the mean recovery of EHDP was 99.9% for 20 analyses (SD. = 3.9). For these determinations, the correlation coefficient was r = 0.9997. Therefore the total phosphorus present in EHDP in this range of concentrations is determined with accuracy as Pi. De termination in urine Accuracy. To check the accuracy of the technique, 43 determinations were performed in which known amounts of EHDP were added to 5 ml of urine from healthy people (concentration range 7.5-300 pmol/l). This urine should have been free of EHDP. The mean t S.E. recovery of EHDP was 100.6 f 0.5 and did not vary with the concentration of EHDP (Table I). The correlation coefficient between the EHDP added and recovered was r = 0.9991. Specificity. With regard to specificity of the method, it must be noted that urines with no EHDP added and with no oral administration of EHDP show a mean content of 1.29 I.tmol/l EHDP (S.D. = 0.738; n = 25). Although this value is below the statistically determined detection limit of the method (2.5 pmol/l, see below), it may be due to phosphorus compounds other than diphosphonates, since there has been no description of the presence in the body of naturally occurring diphosphonates. This value cannot be attributed to either cyclic AMP or to aminoethylphosphonate, a phosphonate found in biological sources, principally in brain, because these two substances added to urine are not measured when urine is analysed as described. However, in patients receiving EHDP at the usual dose of 20 mg/kg body weight daily, the concentration excreted in the urine is 10 to 100 times above this value. Sensitivity. The sensitivity of the method can be improved by taking larger amounts of urine; up to 20 ml or more were tried successfully without

TABLE

I

ACCURACY Recovery of non-radioactive EHDP added to 5 ml of urine or 2 ml of plasma measured by the isotope dilution technique described in the text. EHDP was added at given concentrations within the ranges indicated.

Urine

Plasma

Range of concentrations of EHDP added (pnol/l)

Number of determinations

Mean recovery k S.E.

7.Fr 15 22 - 45 60 -110 150 -300

11 12 10 10

100.4 100.1 101.8 100.0

k f f 2

7.5-300

43

100.6

+ 0.5

1.3 1.1 0.8 1.1

0.52.66.510 14 -

2 2 9 12 20

14 18 13 13 18

90.6 90.9 91.3 94.8 93.9

+ 2.8 f 2.1 + 2.3 -k 2.1 f 0.9

0.5-

20

76

92.3

* 0.9

in %

305

any changes in the method described. With 20 ml of urine and no EHDP administration, the mean content measured as EHDP is about half that found when 5 ml of urine are used. For this reason it is advisable to perform the analysis with 20 ml of urine if it comes from patients receiving a dose of 5 mg/kg/ day, because these patients excrete very low amounts of EHDP. Precision. The precision of the method was checked on 46 determinations in duplicate of urine from patients receiving EHDP. Analyses were done on separate days. The results are summarized in Table II. The standard deviation increases but the coefficient of variation decreases (from 17.7% to 2.3%) as the EHDP concentration increases. The mean coefficient of variation for the whole range (1.3-300 pmol/l) is 3.7%. Detection limit. To determine the detection limit of the method, the variance of the blank, that is of urine without EHDP (S.D. = 0.738 pmol/l) and of the lowest concentration tested (S.D. = 0.16 pmol/l) were taken into account [12]. The detection limit of EHDP was 2.5 pmol/l; to this value the mean content of “EHDP” in urine with no EHDP administration (1.29 pmol/l) has to be added, that is, a result has to be above 3.8 pmol/l to be reliable. Comparison with other techniques. For comparison with other techniques, the EHDP content in 30 urine samples from 2 patients receiving 20 mg EHDP/kg body weight daily by mouth has been determined by this technique and the technique of Liggett [ 71. The results are shown in Fig. 2. The coefficient of correlation was r = 0.902, which indicates a good agreement with no consistent difference between the 2 procedures. The daily excretion of EHDP of patients receiving orally 20 mg EHDP/kg body weight daily lies between 10 and 250 pmol. This methods has been in regular use for more than 2 years during which more than 800 urine determinations have been performed. A single technician can do 6-8 urine analyses daily.

TABLE II REPRODUCIBILITY OF DUPLICATE MEASUREMENTS VARIOUS-CONCENTRATIONS OF EHDP

Urine

Plasma

OF EHDP IN URINE AND IN PLASMA AT

Range of concentrations (~nlOl/l)

Mean of concentrations (jmml/l)

Number of duplicate measurements

S.D. =

Coefficient of variation

1.38 10 - 20 20 - 50 56 -120 130 -300

3.827 15.13 31.73 85.24 196.2

6 9 12 8 11

0.679 0.719 0.841 3.303 4.600

17.7 4.75 2.65 3.87 2.34

1.3-300

73.49

46

2.702

3.68

0.52.56.59 13 -

2 5 8 12 20

1.096 3.742 6.907 10.07 16.19

7 10 7 7 9

0.1065 0.3056 0.6055 0.6426 0.8269

9.72 8.17 8.77 6.38 5.11

0.5-

20

7.741

40

0.5618

7.26

306

Determination in plasma Accuracy. To check the accuracy

of the method, 76 determinations were performed, adding known amounts of EHDP of 2 ml of plasma from healthy people (concentration range 0.5-20 pmol/l) (Table I). The mean recovery f SE. of EHDP for these 76 determinations was 92.3% f 0.9, with a slightly better recovery at larger concentrations. The correlation coefficient between added and recovered concentrations was r = 0.9952. Specificity. Concerning specificity, when plasma without EHDP was analysed, in contrast to urine, the EHDP found was negligible, varying between negative and positive values: 10 plasma samples from healthy people were analysed and gave a mean of 0.036 pmol/l (S.D. = 0.128). The technique therefore seems specific for diphosphonates under the conditions used. Sensitivity. The sensitivity of the method can be improved by taking larger amounts of plasma. The volume of 2 ml was chosen in order to measure EHDP also in small laboratory animals. It should be noted that the amount of [’ “C] EHDP added to 2 ml of plasma for the recovery determination corresponds to a concentration of 0.73 pmol/l. By subtracting this value from the result of the analysis, the error on the differences becomes quite high for the lowest EHDP concentrations; in this case it is therefore advisable to work with 5 ml of plasma. Precision. The precision of the plasma technique was checked on 40 duplicate determinations of 2 ml of plasma analysed on separate days. The results are summarized on Table II. As for urine, the standard deviation increases but the coefficient of variation decreases (from 9.7 to 5.1%) as the EHDP concentration increases. The mean coefficient of variation for the whole range of concentrations (0.5-20 pmol/l) is 7.3%. Detection limit. The detection limit calculated for plasma in the same way as for urine was 0.5 pmol/l EHDP. The EHDP concentration in plasma from patients receiving orally 20 mg

Presented

method

Fig. 2. Correlation between EHDP concentration found in 30 urine samples when determined with the technique described by Liggett [7] end the technique described in this paper.

301

EHDP/kg body weight daily was generally less than taken 1 hour after the oral administration. This technique can theoretically also be used to phonates. It will, however, be necessary to check the recovery of the different precipitations. A method methylenediphosphonate is being developed.

1 pmol/l

if blood

was

measure other diphosUV-photolysis and the to determine dichloro-

Acknowledgements We wish to thank Mrs, S. Miiller and B. Leefe for their technical assistance, Dr. R.G.G. Russell for reviewing the manuscript, and the Procter and Gamble Company for the EHDP determinations in urine according to Liggett. This work has been supported by the Swiss National Research Foundation (No. 3.121.73), the US Public Health Service (No. AM-07266) and the Procter and Gamble Company, Cincinnati, Ohio, U.S.A. References 1 H.A. Fleisch. R.G.G. RusseII. S. Bisaz, R.C. MiihIbauer and D.A. WiIIiams, Eur. J. Clin. Invest., 1 (1970) 12 2 R.G.G. Russell, R.C. MiihIbauer. S.Bisaz. D.A. WiIIiams and H. Fleisch. Chic. Tiss. Res., 6 (1970) 183 3 W.B. Geho and J.A. Whiteside in B. Frame. A.M. Parfitt and H. Duncan teds.). Clinical Aspect-s of Metabolic Bone Disease, Excerpta Medica. Amsterdam, 1973. p. 506 4 R.G.G. Russell, R. Smith, C. Preston, R.J. Walton and C.G. Woods, Lance&i (1974) 894 5 R.D. AItman, C.C. Johnston, M.R.A. Khairi. H. WeIlman, A.N. Serafini and R.R. Sankey, New EngI. J. Med., 289 (1973) 1379 6 J. Guncaga. Th. Lauffenburger, Ch. Lentner. M.A. Dambacher. H.G. Haas. H. Fleisch and A.J. Olah. Harm. Metab. Res., 6 (1974) 62 7 S.J. Liggett. Biochem. Med., 7 (1973) 68 8 A. Jung. S. Bisaz and H. Fleisch. CaIc. Tiss. Res.. 11 (1973) 269 9 Y. Sugino and Y. Miyoshi. J. Biol. Chem.. 239 (1964) 2360 10 S. Bisaz, R.G.G. Russell and H. Fletih, Arch. Oral Biol.. 13 (1968) 683 11 R.A. Libby, Inorg. Chem.. 10 (1971) 386 12 L. Sachs, Angewandte Statistik, Swinger Verlag, Berlin, Heidelberg, New York, 4th edn.. 1973, p. 160