Bone, 7, 255-259 (1986) Printed in the USA. All rights reserved.

8756.3282186 $3.00 + .OO Copyright 0 1986 Pergamon Journals Ltd.

Cellular Phosphate Metabolism in Patients Bisphosphonate Therapy A. CHALLA, A.A. NOORWALI, A. BEVINGTON, and R.G.G. RUSSELL

Receiving

Departfnent of kkur?an Metabolwn & Clinical BiochemMry, Unwerslty of Sheffield MedIcal School, Sheffield, UK

Address for correspondence and reprints: Dr. A. Bevington, Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield St0 2RX, U.K

Abstract

Patients with Paget’s disease of bone were treated with oral disodium dihydrogen ethylidene-l-hydroxy-l,l- bisphosphonate (EHBP), a drug that is known to stimu- late renal tubular reabsorption of orthophosphate (Pi). After 2 weeks of treatment, plasma Pi rose from 1.02 to 1.67 mmol/l. No increase in Pi was observed with the related drug, dichloromethylene bisphosphonate, which also reduces bone turnover in Paget’s disease. Intravenous EHBP caused a more rapid increase in plasma Pi, but maximum hyperphosphatemia was not observed until 7-l 1 days after treatment commenced. It is therefore unlikely that this effect is due to an imme- diate action of EHBP on the luminal face of the renal brush border Pi transporter. After 2 weeks of oral EHBP, the increase in the Pi concentration in patients’ erythrocytes was 31% compared with 64% in plasma. In blood platelets and leukocytes, negligible changes in cellular Pi occurred. The concentrations of 2,3-diphos- phoglycerate, adenosine 5’-diphosphate (ADP) and adenosine 5’-triphosphate (ATP) were unaltered, indi- cating that these organic phosphates were not offset- ting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also ob- served in patients receiving intravenous EHBP. How- ever, no change occurred in cell/plasma distribution of chloride, suggesting that this apparent regulation of cellular P, did not arise from changes in erythrocyte membrane potential, pH, or water content.

Key Words: Phosphates-Bisphosphonates-Diphos- phonates-Erythrocytes-Blood Platelets-Leukocytes.

Introduction

In man and other mammals the extracellular concentration of orthophosphate (P,) is regulated largely by the kidney by alterations in the renal tubular reabsorption of phos- phate (Massry and Fleisch, 1980). Although several factors that influence the renal handling of phosphate are known, such as parathyroid hormone and growth hor- mone, much remains to be learned about the control mechanism. For example, the mechanisms underlying the renal conservation of phosphate during phosphate depri- vation are not known. One of the most impressive drug-in-

duced changes in phosphate metabolism occurs with the bisphosphonate drug ethylidene-I-hydroxy-I ,l-bisphos- phonate (EHBP), which can markedly stimulate renal tu- bular reabsorption of P, in humans, leading to hyperphos- phatemia (Walton et al., 1975). This effect cannot be ex- plained by changes in any of the hormones known to affect renal P, handling (Walton et al., 1974).

Studies on this effect in humans have been restricted largely to work on blood serum and urine, and little is known of the corresponding intracellular changes. In this study, we have attempted to relate the changes in extra- cellular P, to the cellular concentrations of P, and organic phosphates in readily accessible human cells (erythro- cytes, blood platelets, and leukocytes) from patients with Paget’s disease of bone before and after treatment with oral EHBP. In addition, we have studied the time course of these effects in greater detail in patients receiving intrave- nous EHBP, thus bypassing intestinal absorption, which may have been a rate-limiting factor in earlier studies.

Preliminary accounts of this work have been presented (Preston et al., 1982; Bevington et al., 1983).

Materials and Methods

Venous blood samples (10 ml) were drawn Into heparin from pa- tients after an overnight fast and immediately chilled on ice. Phos- phorus metabolites were measured in erythrocytes from 10 pa- tients receiving oral EHBP, 5 patients receiving intravenous EHBP and 18 patients receiving oral dichloromethylene-bisphosphonate (CI,MBP). Doses and duration of treatment are described in the tables and fiaure leaend. Of the 10 Datients receivina oral EHBP. 4 were taken-at randvom for determination of phosph&us metabo- lites in their leukocytes and blood platelets.

Erythrocytes

Heparinized blood or plasma samples (0.5 ml) were pipetted into 1 0 ml ice-cold 1 molil perchloric acid, 15-30 min after the blood was drawn, and centrifbged at 3000 g for 15 min at 4°C to remove precipitated protein The supernatants were adjusted to pH 5-7 by addition of 4.3 molil potassium hydroxide containing 0.6 mol/l imidazole.

P, assay

The neutralized extract (0 2 ml) was mixed with 0.2 ml of 27 mmolil ammonium molybdate [(NH,),Mo,0Z4. 4H,O] in 2.4 mol/l hydrochloric acid This was followed immediately by addition of 0.4 ml of a solution containing 1 vol of petroleum spirit (boiling

255

256

point 80-100°C) and 4 vol of 2-methylpropan-lo. This mixture was then shaken for 10 s to extract phosphomolybdate into the organic solvent phase. The aqueous and organic phases were separated by centrifugation at 1000 g for 1 min at 4”C, and 0.25 ml of the upper (organic) phase was mixed with 2.5 ml of ethanol, followed by 0.025 ml of freshly prepared 0.18 molil SnCI, . 2H,O in 1.65 mol/l hydrochloric acid. After 10 min, the absorbance was read at 725 nm.

Erythrocyte P, concentration (C) was calculated from the ex- pression:

c= 1008 - (100 - PCV)P

mmolil cells PCV

where 6 is the concentration in blood, P the concentration in plasma, and PCV the percentage packed cell volume of the blood. A similar calculation was used for platelet and leukocyte suspensions.

For internally standardized measurements, the perchloric acid used to deproteinize the samples contained 0 48 mmol Na2HP0,/ I, so that the amount of P, added was comparable to that in normal blood (Challa et al., 1985).

Platelets were isolated from 10 ml blood plus 1 1 ml 0.13 molil sodium citrate (Na,C,H,O, * 2H,O) by centrifuging at 150 g for 10 min at 4°C removing the supernatant, and recentrifuging the su- pernatant at 1500 g for 10 mm at 4°C to sediment the platelets

Leukocytes were obtained by mixing 3 vol of the infernatant from the 150 g centrifugation with 1 vol of 6% w/v dextran [average molecular weight 256,000 (Sigma)], In 154 mmolil NaCI. This was allowed to sediment for 45 min at 4°C under gravity The supernatant was then centrifuged at 800 g for 5 min at 4°C to sediment the leukocytes. Samples (0.5 ml) of platelet or leukocyte suspension, titrated plasma, or titrated plasmaidextran were then deproteinized and assayed for P, as described for the eryth- rocytes

Cell water volume in platelets and leukocytes

Separate 0.5 ml samples of the suspensions were incubated for 10 min at 37°C with 9.3 kBq (0.25 &i) 3H,0 (Amersham TRS 3) and 1.9 kBq (0.05 f&i) inulin-[14C] carboxyltc acid (Amersham CFA 399). The cells were sedimented at 1500 g for 10 min at room temperature and resuspended in 0.5 ml 154 mmolil NaCI. Samples (0.1 ml) were taken for dual isotope liquid scintillation counting. Counting efficiency was assessed by adding known amounts of 3H,0 or inulin-[r4C] carboxylic acid to previously unla- beled cell suspensions lust prior to counting 3H and i4C counts from the cell pellets were corrected for counting efficiency, and cellular water volume was calculated by assuming that 3H, but not 14C, had free access to the intracellular space (Casey et al.. 1977)

Organic phosphates and chloride were measured in the neu- tralized perchlorate extracts described above Adenosine 5’.tri- phosphate (ATP) and adenosine 5’-diphosphate (ADP) were sep- arated on a Waters Associates Radial-Pak C-18 10~ reverse phase column, eluting with 0.125 mol/l ammonium phosphate, pH 5 at 1.5 ml/min, and were detected on a Waters Model 440 Absor-

A Challa et al Brsphosphonates and cellular phosphate

bance Detector at 254 nm. Elution times were typrcally 6 0 mm for ATP and 7.2 min for ADP. Standards containing known concen- trations of the sodium salts of the nucleotides (Sigma) were pre- pared by addition of perchloric acid, potassium hydroxide, and imldazole as described for the cell extracts. Calibration curves were constructed by plotting the peak height detected for each nucleotide against the concentration of the standards Similar re- sults were obtained if peak areas were used in the calibration In place of peak heights. 2,3-Diphosphoglycerate (2,3-DPG) was assayed as P, after hydrolysis in the presence of phospho- glycerate mutase (Sigma, 1975). Chloride was measured colorr- metrically as described by Zall et al. (1956) The renal tubular reabsorption of P,, expressed as T, ,,IGFR, was measured as de- scribed by Walton and Bijvoet (1977)

Drugs

Disodium dihydrogen EHBP [also known as etldronate disodrum (EHDP) or Didronel (IV)] and disodium dihydrogen CI,MBP (Clo- dronate) were obtained from Proctor & Gamble Co, Cincinnati, Ohio. USA

Statistical analysis

Statistical significance of changes was assessed by parred Stu- dent’s t-test.

Results

In agreement with earlier studies (Walton et al., 1975) 2 weeks of oral therapy with EHBP caused a significant in- crease (64%) In the plasma concentration of P, (Table I). However, the corresponding concentration of P, in erythro- cytes rose by only 31%. Hence the erythrocyteiplasma distribution ratio for P, showed a small, but significant de- crease (Table I). A markedly different result occurred in patients receiving the closely related drug CI,MBP. No srg- nificant changes in plasma or erythrocyte concentrations of P, were observed either after 2 weeks (Table I) or after periods as long as 28 weeks of this treatment.

Concentrations of ATP and 2,3-DPG in the erythrocytes of patients receiving EHBP or CI,MBP showed no signifi- cant change (Table II), even in those who became hyper- phosphatemic during treatment with EHBP.

When an additional 5 patients with Paget’s drsease were given a 5day course of intravenous EHBP, the plasma concentration of P, and T,,,/GFR rose during treat- ment but only reached maximum values 7-I 1 days after the first infusion of the drug (Table Ill). As in the pa- tients who received oral EHBP (Table I), the corresponding erythrocyteiplasma distribution ratio for P, decreased during hyperphosphatemia (Table Ill). No change in eryth-

Table I. The effect of oral bisphosphonates on the distribution of P, between erythrocytes and plasma.

EHBP” C12MBPa

Pretreatment After 2 Weeks Pretreatment After 2 Weeks

No. of patients 10 10 18 18 Plasma P, (mmol/l plasma) 1.02 + 0.26 1 67b 2 0.33 1 09 * 0.13 100 & 013 Erythrocyte P,

(mmolil packed cells) 0.71 2 0.21 0.93c -+ 0 22 0.62 ? 0.23 0.54 ? 0.24 Erythrocyteiplasma P, ratio 0.70 2 0.16 0.56d ? 0.15 0.57 2 0.23 0.54 t 0.28

a All values are mean ? 1 standard deviatron b P < 0.001 relative to Pretreatment value c P < 0.01 relative to Pretreatment value d P < 0.05 relative to Pretreatment value. Patients with Paget’s disease received either 1600 mg (6 4 mmol) of EHBP per day or 1600 mg (5.5 mmol) CI,MBP per day.

A. Challa et al.: Bisphosphonates and cellular phosphate 257

Table II. Aqueous organic phosphate concentrations in erythrocytes, blood platelets, and leukocytes of patients receiving oral bisphos- phonates.

Pretreatment

Erythrocytesa,b

EHBP CI,MBP

After 2 Weeks Pretreatment After 2 Weeks

No. of patients ATP (mmolil packed cells) 2,3-DPG (mmolil

packed cells)

No. of patients ATP (mmol/l cell water) No. of patients ADP (mmol/l cell water)

10 10 1.37 2 0.24 1.50 c 0 24

4.85 f 1.05 457 -t 1.60

Piateletsa,b

Pretreatment After 2 Weeks

4 4 2.49 * 0.29 2 52 t 0.32

4 4 1.52 2 0.12 1 70 t 0.16

18 18 1.51 ” 0.32 145 f 031

4.14 -r- 047 3.92 * 1.32

Leukocyte@

Pretreatment After 2 Weeks

4 4 199 * 015 219 2 025

3 3 1.05 + 0 10 0.94 ? 0 10

a All values are mean 2 1 standard deviation. b Patients with Paget’s disease received either 1600 mg (6 4 mmol) of EHBP per day or 1600 mg (5.5 mmol) of CI,MBP per day. Erythrocyte values were measured In the same cell extracts as the values in Table I. Platelet and leukocyte values were measured in the same cell extracts as Figure 1

rocyte/plasma chloride ion distribution was detected in the same blood samples.

A more marked effect on cell/plasma P, distribution was observed in blood platelets and leukocytes in 4 hyper-

phosphatemic patients who received oral EHBP. In these cells the observed increase in the concentration of P, was negligible in relation to that in plasma (Fig. 1). However, as in erythrocytes, the intracellular concentration of ATP did not change significantly in platelets and leukocytes as a result of hyperphosphatemia. There was also no marked change observed in the concentration of ADP (Table II).

Discussion

In patients receiving high oral doses of EHBP (Walton et al., 1975; and Table I), the rate-limiting factor in the rise of T,,,/GFR and plasma P, concentration could simply be in- testrnal absorption of the drug. Bypassing the intestine by administration of EHBP Intravenously (Table Ill) led to changes of similar magnitude in a matter of days, and these changes persisted for several days after treatment had ceased. A similar observation has been reported in

dogs (Francis and Slough, 1984), in which hyperphospha- temia was maintained for several days after infusion of the drug even though circulating EHBP concentrations had fallen to negligible levels. At the high doses used in these animal studies renal impairment may also have contrib- uted to the hyperphosphatemia (Bounameaux et al., 1983). This, however, is unlikely to be an important factor under the conditions used in humans (Kanis et al., 1983).

These results seem inconsistent with a direct action of the drug on the actrve P, transport mechanism at the Iu- minal surface of the renal brush border membrane (Kessler and Fanestil, 1981) and suggest that an addi- tional rate-limiting process is required before changes in tubular P, transport occur. For example, it has been sug- gested that, in rats, the effect of high doses of EHBP on renal Pi handling arises indirectly as a response of the kidney to blocking of bone mineralization by the drug (Bonjour et al., 1978; Stall et al., 1980). However, the bio- chemical mechanism of this putative coupling between mineralization and renal P, handling is still unclear, and its relevance to the stimulation of T,,,IGFR by EHBP in humans is uncertain, as the high doses of EHBP used in

Table Ill. Comparison of the distribution of P, and chloride between erythrocytes and plasma during treatment with intravenous EHBP.

Plasma P, (mmolil plasma)”

T,,,/GFRa (mmolll glomerular

filtrate (n = 4)

Erythrocyteiplasma P, Ratioa

(mmolil packed cells)

(mmolil plasma)

ErythrocyteiPlasma Chloride Ratioa

(mmolil packed cells)

(mmol/l plasma)

Pretreatment 115 * 011 1.06 2 0.16 0.61 + 0 08 0 60 lr 0.07 Posttreatment 2 28b 2 0.24 2.40c ? 0 38 0.39d * 0 09 0.60 -t 0.06

a All values are mean ?Z 1 standard deviation. b P < 0.005 relative to Pretreatment value. c P < 0 02 relative to Pretreatment value d P < 0.05 relative to Pretreatment value. Frve patients with Paget’s disease received a continuous infusion of 300 mg (1 2 mmol) Intravenous EHBP from 10.00 to 13 00 h each day for 5 successive days. Pretreatment values were sampled just before the first infusion. Posttreatment values refer to the day on whrch maximum hyperphosphatemia was observed, whrch was 7--l 1 days (mean 9 days) after the first infusion. All plasma and erythrocyte P, concentrations in this table were measured in an Internally standardized assay

A Challa et al Bisphosphonates and cellular phosphate

Plasma Erythrocytes Platelets Leukocytes

OL- OL- CJ- - d- - Off On Off On Off On Off On

Fig. 1. Comparison of the responses of cellular P, to hyperphos- phatemia induced by EHBP in erythrocytes, platelets, and leuko- cytes. Blood samples were drawn from 4 patients with Paget’s disease before treatment (Off) and after 2 weeks of oral EHBP at 1600 mg (6.4 mmol) per day (On). P, was measured in all three cell types in the same blood sample Plasma P, is expressed in mmolil plasma, erythrocyte P, in mmolil packed cells, and platelet and leukocyte P, in mmolil cell water The percentage Increase in plasma P, was 86 ? 56% (mean ? 1 standard devration). In a paired comparison, the percentage increase in cellular P, was sig- nificantly less than that in plasma for all three cell types: erythro- cytes 34 * 11% (P < 0.05) platelets 5 3 c 2.2% (P < 0 OOl), leukocytes 9.2 2 4 9% (P < 0.002)

the rat experiments lead to phosphaturia rather than in- creased Pi retention.

In an earlier study (Challa et al., 1985), we showed that in severely hypophosphatemic or hyperphosphatemic pa- tients in an intensive care unit the erythrocyte P, concen- trations were roughly proportional to those in plasma. These observations are consistent with passive distribu- tion of P, across the cell membrane being the dominant factor determining the cellular P, concentration. In con- trast, in the present study changes in cellular P, during treatment with EHBP were small in comparison with those in plasma P,, especially in blood platelets and leukocytes (Fig. 1). This did not arise from an effect of the drug on the P, assay, since similar results were obtained by internally standardized measurements (Table Ill). However, the pro- portionality observed in the earlier work (Challa et al., 1985) was heavily dependent on samples from patients whose plasma concentration of P, was either extremely high (above 2.0 mmolil) or extremely low (below 0.5 mmoli I). Over the intermediate range (which encompasses that in the present study) wide variation in erythrocyte P, con- centration was observed, so that no firm conclusion could be drawn about the relationship between erythrocyte P, and plasma P, over this range (Challa et al., 1985). There- fore, it is possible that in the earlier work a phenomenon like that reported in the present study did occur but was simply obscured by greater biologic variation.

Prolonged in vitro exposure of cultured calvaria cells to EHBP has no detectable effect on the amount of P, in the cells (Felix and Fleisch, 1982), and in the present study we detected no simple physical or chemical changes in the patients’ blood cells that might have arisen from a direct action of the drug. For example, in erythrocytes, drug-in- duced changes in the membrane potential, pH, or water content could alter the distribution of permeant anions, in- cluding P,, across the cell membrane (Hladky and Rink,

1977; but we showed that these factors were probably un- important here, as the distribution of the passively trans- ported anion Cl- was unaltered (Table Ill). Alternatively. if EHBP caused a continuous flux of P, into aqueous organrc phosphate pools, this could buffer the cellular P, concen- tration at a lower value than that predicted from the In- crease In plasma P,. This also was shown to be unlikely, as the size of the largest aqueous pools (2,3-DPG, ATP, and ADP), was unaltered in the three cell types, even after 2 weeks of oral EHBP. However, this does not rule out the possibility of buffering of intracellular P, concentratrons by phospholrpid pools by a process analogous to that re- ported in plasma (Miihlbauer and Fleisch, 1985).

These findings are consistent with earlier work that sug- gested that there was no increase in the pool size for P, outside the extracellular compartment during Infusions of phosphate (Walton et al., 1975), although direct measure- ments of the largest intracellular P, pools (e.g., those in skeletal muscle) have not yet been reported. Ditzel et al. (1977), who gave EHBP in an attempt to raise 2,3-DPG in erythrocytes and thereby potentially improve tissue oxy- genation in patients with diabetes mellitus, also failed to detect significant changes in intracellular 2,3-DPG In spite of substantial change in extracellular P,, in patients given doses of EHBP comparable to those given here.

These anomalously small changes in cellular P, suggest that cellular concentrations of this ion cannot be explained solely by passive distribution across the plasma mem- brane. This is consistent with the observation that, at normal extracellular P, concentrations (1 mmolil), erythro- cytes at 37°C In vitro have a cellular P, concentration higher than that predicted for passively distributed HPO,*- and H,PO,- ions (Challa et al., 1985). Similarly, at lower concentrations of plasma P, (0.65 mmolil) in patients with vitamin D-resistant rickets, 31P clinical magnetic reso- nance measurements suggest that P, concentrations are normal in the patients’ skeletal muscle (Smith et al., 1984) Measurements on acid extracts of renal cortex have led to a similar conclusion in hypophosphatemic mice (Brown et al., 1985).

It will be interesting to see whether this apparent resrs- tance to change by cellular P, occurs in other forms of hy- pophosphatemia and hyperphosphatemia and whether it is a true regulatory mechanism or an indirect conse- quence of the patient’s illness or therapy.

Achnowledgement The financial support of the Rehabilitation and Medical Research Trust IS acknowledged. A.B was supported by a J.G. Graves Medical Research Fellowship from the University of Sheffield

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Received, September 10, 1985

Reused January 30, 1986

Accepted March 7. 1986