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Cellular phosphate metabolism in patients receiving bisphosphonate therapy
Bone, 7, 255-259 (1986) Printed in the USA. All rights reserved.
8756.3282186 $3.00 + .OO 0 1986 Pergamon Journals Ltd.
Copyright
Cellular Phosphate Metabolism in Patients Receiving Bisphosphonate Therapy A. CHALLA,
A.A. NOORWALI,
A. BEVINGTON,
and R.G.G. RUSSELL
Departfnent of kkur?anMetabolwn & Clinical BiochemMry, Unwerslty of Sheffield MedIcal School, Sheffield, UK Address for correspondence and reprints: Dr. A. Bevington, Sheffield Medical
Department School, Beech Hill Road, Sheffield St0 2RX, U.K
Abstract
of Human Metabolism
& Clinical Biochemistry,
University
of
duced changes in phosphate metabolism occurs with the bisphosphonate drug ethylidene-I-hydroxy-I ,l-bisphosphonate (EHBP), which can markedly stimulate renal tubular reabsorption of P, in humans, leading to hyperphosphatemia (Walton et al., 1975). This effect cannot be explained 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 extracellular P, to the cellular concentrations of P, and organic phosphates in readily accessible human cells (erythrocytes, 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 intravenous 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).
Patients with Paget’s disease of bone were treated with oral disodium dihydrogen ethylidene-l-hydroxy-l,lbisphosphonate (EHBP), a drug that is known to stimulate 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 immediate 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-diphosphoglycerate, adenosine 5’-diphosphate (ADP) and adenosine 5’-triphosphate (ATP) were unaltered, indicating that these organic phosphates were not offsetting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also observed in patients receiving intravenous EHBP. However, 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.
Materials and Methods Venous blood samples (10 ml) were drawn Into heparin from patients after an overnight fast and immediately chilled on ice. Phosphorus metabolites were measured in erythrocytes from 10 patients 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.
Key Words:
Phosphates-Bisphosphonates-Diphosphonates-Erythrocytes-Blood Platelets-Leukocytes.
4 were taken-at randvom for determination of phosph&us lites in their leukocytes and blood platelets.
metabo-
Erythrocytes
Introduction
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.
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 phosphate (Massry and Fleisch, 1980). Although several factors that influence the renal handling of phosphate are known, such as parathyroid hormone and growth hormone, much remains to be learned about the control mechanism. For example, the mechanisms underlying the renal conservation of phosphate during phosphate deprivation are not known. One of the most impressive drug-in-
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
A Challa et al
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 expression: 1008 - (100 -
PCV)P mmolil cells
c= 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 supernatant 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 erythrocytes 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 unlabeled 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 neutralized perchlorate extracts described above Adenosine 5’.triphosphate (ATP) and adenosine 5’-diphosphate (ADP) were separated 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-
Table I. The effect of oral bisphosphonates
on the distribution
Brsphosphonates
bance Detector at 254 nm. Elution times were typrcally 6 0 mm for ATP and 7.2 min for ADP. Standards containing known concentrations of the sodium salts of the nucleotides (Sigma) were prepared 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 results 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 phosphoglycerate mutase (Sigma, 1975). Chloride was measured colorrmetrically as described by Zall et al. (1956) The renal tubular reabsorption of P,, expressed as T, ,,IGFR, was measured as described by Walton and Bijvoet (1977)
Drugs Disodium dihydrogen EHBP [also known as etldronate disodrum (EHDP) or Didronel (IV)] and disodium dihydrogen CI,MBP (Clodronate) were obtained from Proctor & Gamble Co, Cincinnati,
Ohio. USA Statistical
analysis
Statistical significance dent’s t-test.
of changes
was assessed
by parred Stu-
Results In agreement with earlier studies (Walton et al., 1975) 2 weeks of oral therapy with EHBP caused a significant increase (64%) In the plasma concentration of P, (Table I). However, the corresponding concentration of P, in erythrocytes rose by only 31%. Hence the erythrocyteiplasma distribution ratio for P, showed a small, but significant decrease (Table I). A markedly different result occurred in patients receiving the closely related drug CI,MBP. No srgnificant 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 significant change (Table II), even in those who became hyperphosphatemic 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 treatment but only reached maximum values 7-I 1 days after the first infusion of the drug (Table Ill). As in the patients who received oral EHBP (Table I), the corresponding erythrocyteiplasma distribution ratio for P, decreased during hyperphosphatemia (Table Ill). No change in eryth-
of P, between erythrocytes
and plasma.
EHBP”
No. of patients Plasma P, (mmol/l plasma) Erythrocyte P, (mmolil packed cells) Erythrocyteiplasma P, ratio
and cellular phosphate
C12MBPa
Pretreatment
After 2 Weeks
Pretreatment
After 2 Weeks
10 1.02 + 0.26
10 1 67b 2 0.33
18 1 09 * 0.13
18 100 & 013
0.71 2 0.21 0.70 2 0.16
0.93c -+ 0 22 0.56d ? 0.15
0.62 ? 0.23 0.57 2 0.23
0.54 ? 0.24 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
257
and cellular phosphate
Table II. Aqueous organic phosphate
concentrations
in erythrocytes,
blood platelets, and leukocytes
of patients receiving oral bisphos-
phonates. Erythrocytesa,b CI,MBP
EHBP
No. of patients ATP (mmolil packed 2,3-DPG (mmolil
cells)
packed cells)
After 2 Weeks
Pretreatment
After 2 Weeks
Pretreatment
10 1.37 2 0.24
10 1.50 c 0 24
18 1.51 ” 0.32
145
4.85 f
457
4.14 -r- 047
3.92 * 1.32
1.05
-t 1.60
Leukocyte@
Piateletsa,b
No. of patients ATP (mmol/l cell water) No. of patients ADP (mmol/l cell water)
18 f 031
Pretreatment
After 2 Weeks
4 2.49 * 0.29 4 1.52 2 0.12
4 2 52 t 0.32 4 1 70 t 0.16
Pretreatment
After 2 Weeks
4 199 * 015 3 1.05 + 0 10
4 2 025 3 0.94 ? 0 10
219
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 hyperphosphatemic 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 intestrnal 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
Table Ill. Comparison
Pretreatment Posttreatment
of the distribution
of P, and chloride
dogs (Francis and Slough, 1984), in which hyperphosphatemia 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 contributed 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 Iuminal surface of the renal brush border membrane (Kessler and Fanestil, 1981) and suggest that an additional rate-limiting process is required before changes in tubular P, transport occur. For example, it has been suggested 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 biochemical 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
between erythrocytes
Plasma P, (mmolil plasma)”
T,,,/GFRa (mmolll glomerular filtrate (n = 4)
115 * 011 2 28b 2 0.24
1.06 2 0.16 2.40c ? 0 38
and plasma during treatment
Erythrocyteiplasma P, Ratioa (mmolil packed cells)
with intravenous
ErythrocyteiPlasma Ratioa (mmolil packed
EHBP. Chloride cells)
(mmolil plasma)
(mmol/l plasma)
0.61 + 0 08 0.39d * 0 09
0 60 lr 0.07 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
OLOff
Platelets
Erythrocytes
Plasma
OLOn
Off
Leukocytes
d- -
CJOn
Off
On
Off
On
Fig. 1. Comparison
of the responses of cellular P, to hyperphosphatemia induced by EHBP in erythrocytes, platelets, and leukocytes. 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 significantly less than that in plasma for all three cell types: erythrocytes 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 increased Pi retention. In an earlier study (Challa et al., 1985), we showed that in severely hypophosphatemic or hyperphosphatemic patients in an intensive care unit the erythrocyte P, concentrations were roughly proportional to those in plasma. These observations are consistent with passive distribution of P, across the cell membrane being the dominant factor determining the cellular P, concentration. In contrast, 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 proportionality 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, concentration 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). Therefore, 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-induced changes in the membrane potential, pH, or water content could alter the distribution of permeant anions, including P,, across the cell membrane (Hladky and Rink,
Bisphosphonates
and cellular phosphate
1977; but we showed that these factors were probably unimportant here, as the distribution of the passively transported 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, concentration at a lower value than that predicted from the Increase 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 reported in plasma (Miihlbauer and Fleisch, 1985). These findings are consistent with earlier work that suggested 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 measurements 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 oxygenation 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 membrane. This is consistent with the observation that, at normal extracellular P, concentrations (1 mmolil), erythrocytes 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 resonance 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 resrstance to change by cellular P, occurs in other forms of hypophosphatemia and hyperphosphatemia and whether it is a true regulatory mechanism or an indirect consequence 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
References Bevrngton A , Preston C J Challa A, Noorwali A, Kanls J A and Russell R G G. Why does sodium etidronate (EHDP) change the dlstrlbutlon of orthophosphate (P,) across the red cell7 Caicif ~rssue Int 35[Suppl] A37. 1983 Bon)our J -P , Troehler U Preston C and Fleisch H Parathyrold hormone and renal handling of P, Effect of dretary P, and diphosphonates Am J Phys/o/ 234.F497LF505, 1978 Bounameaux H M Schifferll J Montarn J-P, Jung A and Chatelanat F Renal failure assocrated with Intravenous diphosphonates Lancef 1471, 1983 Brown C.E , Wilkie C A Meyer M H and Meyer R A Jr Response of trssue phosphate content to acute dietary phosphate deprlvatron in the Xlinked hypophosphatemic mouse C&/f Trssue Int. 37 4233430, 1985
A. Challa et al.: Bisphosphonates
Casey R P
and cellular phosphate
NJUS D , Radda G K and Sehr P.A.: Active proton uptake by
chromaffin
granules
Observatron
by amine distributron
and phos-
phorus-31 nuclear magnetic resonance techniques Brochemistry 16 972-977, 1977 Challa A, Bevington A, Angier C M Asbury A.J., Preston C J and Russell R G G : A technique for the measurement of orthophosphate in human erythrocytes. and some studies of its determinants C/in SC/ 69 4299434, 1985. Ditzel J Hat. C and Daugaard N Effect of the diphosphonate EHDP on plasma inorganic phosphate and hemoglobin oxygen affinity of diabetic and healthy subjects In Phosphate Metabohsm, Advances ,n Exper/mental Medicine and Biology S G Massry and E Ritz, eds Plenum Press, New York, 1977, Vol 81 Felrx R and Fleisch H Effect of diphosphonates on ATP and P, content, P, uptake and energy charge of cultured calvaria cells Expenentla 38.6444646, 1982 Francis M D. and Slough C L. Acute Intravenous infusion of disodrum dihydrogen (I-hydroxyethylidene)diphosphonate. Mechanrsm of toxicity d. &arm. So. 73 1097- 1100, 1984 Hladky SB and Rink T J.. pH equiltbrrum across the red cell membrane In Membrane Transport In Red Ceils. J C. Ellory and V L Lew, eds Academic Press, London, 1977 Kanrs J.A , Preston C J Yates A J P , Percival R.C , Mundy K I and Russell R G G Effects of Intravenous diphosphonates on renal function iancet 1 1328, 1983 Kessler R J and Fanestrl D D Identification of a phosphate-binding proteolrprd in kidney brush border: In Caicum and Phosphate Transport across &omembranes F Bronner and M Peterlik. eds Academic Press, New York, 1981 Massry S G and Fleisch H (eds) Renal Handling of Phosphate Plenum Publrshlng Corporatron. New York, 1980. Muhlbauer R C. and Fielsch H Inverse relatron between Inorganic phosphate and phospholipids in plasma of mace Min. Elect. Metab. 11 332, 1985
259
Preston C.J
, Noorwali
A., Challa A., Paterson A D
and Russell R G G. Intracellular
Beard D J
Kanrs J A
inorganic phosphate and ATP levels In
human blood erythrocytes, leucocytes and platelets in normal subjects and In diseases assocrated wrth altered phosphate metabolism In Regulabon of Phosphate and Mlnerai MetaboGsm, Advances In Expenmental Medfcfne and Biology. SG Massry, J M Letter1 and E Ritz, eds Plenum Press, New York, 1982, Vol 151 Sgma Technical Bulletrn No 665 The colorrmetrrc enzymatic determrnatron of 2,3-drphosphoglycenc acid Sigma Chemrcal Company, Saint Louis, MO, USA, 1975 Smith R., Newman R J Radda G.K Stokes M and Young A Hypophosphataemrc osteomalacia and myopathy Studies with nuclear magnetic resonance spectroscopy C//n Sci 67 5055509, 1984 Stall R Murer H Fleisch H and Bonfour J-P Effect of dlphosphonate treatment on phosphate transport by renal brush border vesicles Am J Physfol. 239:F13-F16. 1980 Walton R J and Brfvoet 0 L M A sample slide-rule method for the assessment of renal tubular reabsorption of phosphate In man Cl/n Chm Acta 01 273-276, 1977 Walton R J Russell R G G and Smith R Changes rn the renal and extrarenal handling of phosphate induced by disodrum etrdronate (EHDP) rn man C/in So. MO/. Med 49 45-56, 1975 Walton R J Russell R G.G Albert1 K G M M Clark M , Potts J T Bisaz S and Flelsch H : Studies on the hyperphosphataemra induced by drsodrum ethane-l-hydroxy-I ,I-diphosphonate (EHDP) rn man Eur j C//n Invest. 41337, 1974 Zall D M Fisher D and Garner M Q Photometrrc determination of chlorides In water Anal. Chem. 28: 1665 1668, 1956
Received, September 10, 1985 Reused January 30, 1986 Accepted March 7. 1986
8756.3282186 $3.00 + .OO 0 1986 Pergamon Journals Ltd.
Copyright
Cellular Phosphate Metabolism in Patients Receiving Bisphosphonate Therapy A. CHALLA,
A.A. NOORWALI,
A. BEVINGTON,
and R.G.G. RUSSELL
Departfnent of kkur?anMetabolwn & Clinical BiochemMry, Unwerslty of Sheffield MedIcal School, Sheffield, UK Address for correspondence and reprints: Dr. A. Bevington, Sheffield Medical
Department School, Beech Hill Road, Sheffield St0 2RX, U.K
Abstract
of Human Metabolism
& Clinical Biochemistry,
University
of
duced changes in phosphate metabolism occurs with the bisphosphonate drug ethylidene-I-hydroxy-I ,l-bisphosphonate (EHBP), which can markedly stimulate renal tubular reabsorption of P, in humans, leading to hyperphosphatemia (Walton et al., 1975). This effect cannot be explained 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 extracellular P, to the cellular concentrations of P, and organic phosphates in readily accessible human cells (erythrocytes, 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 intravenous 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).
Patients with Paget’s disease of bone were treated with oral disodium dihydrogen ethylidene-l-hydroxy-l,lbisphosphonate (EHBP), a drug that is known to stimulate 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 immediate 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-diphosphoglycerate, adenosine 5’-diphosphate (ADP) and adenosine 5’-triphosphate (ATP) were unaltered, indicating that these organic phosphates were not offsetting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also observed in patients receiving intravenous EHBP. However, 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.
Materials and Methods Venous blood samples (10 ml) were drawn Into heparin from patients after an overnight fast and immediately chilled on ice. Phosphorus metabolites were measured in erythrocytes from 10 patients 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.
Key Words:
Phosphates-Bisphosphonates-Diphosphonates-Erythrocytes-Blood Platelets-Leukocytes.
4 were taken-at randvom for determination of phosph&us lites in their leukocytes and blood platelets.
metabo-
Erythrocytes
Introduction
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.
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 phosphate (Massry and Fleisch, 1980). Although several factors that influence the renal handling of phosphate are known, such as parathyroid hormone and growth hormone, much remains to be learned about the control mechanism. For example, the mechanisms underlying the renal conservation of phosphate during phosphate deprivation are not known. One of the most impressive drug-in-
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
A Challa et al
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 expression: 1008 - (100 -
PCV)P mmolil cells
c= 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 supernatant 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 erythrocytes 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 unlabeled 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 neutralized perchlorate extracts described above Adenosine 5’.triphosphate (ATP) and adenosine 5’-diphosphate (ADP) were separated 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-
Table I. The effect of oral bisphosphonates
on the distribution
Brsphosphonates
bance Detector at 254 nm. Elution times were typrcally 6 0 mm for ATP and 7.2 min for ADP. Standards containing known concentrations of the sodium salts of the nucleotides (Sigma) were prepared 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 results 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 phosphoglycerate mutase (Sigma, 1975). Chloride was measured colorrmetrically as described by Zall et al. (1956) The renal tubular reabsorption of P,, expressed as T, ,,IGFR, was measured as described by Walton and Bijvoet (1977)
Drugs Disodium dihydrogen EHBP [also known as etldronate disodrum (EHDP) or Didronel (IV)] and disodium dihydrogen CI,MBP (Clodronate) were obtained from Proctor & Gamble Co, Cincinnati,
Ohio. USA Statistical
analysis
Statistical significance dent’s t-test.
of changes
was assessed
by parred Stu-
Results In agreement with earlier studies (Walton et al., 1975) 2 weeks of oral therapy with EHBP caused a significant increase (64%) In the plasma concentration of P, (Table I). However, the corresponding concentration of P, in erythrocytes rose by only 31%. Hence the erythrocyteiplasma distribution ratio for P, showed a small, but significant decrease (Table I). A markedly different result occurred in patients receiving the closely related drug CI,MBP. No srgnificant 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 significant change (Table II), even in those who became hyperphosphatemic 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 treatment but only reached maximum values 7-I 1 days after the first infusion of the drug (Table Ill). As in the patients who received oral EHBP (Table I), the corresponding erythrocyteiplasma distribution ratio for P, decreased during hyperphosphatemia (Table Ill). No change in eryth-
of P, between erythrocytes
and plasma.
EHBP”
No. of patients Plasma P, (mmol/l plasma) Erythrocyte P, (mmolil packed cells) Erythrocyteiplasma P, ratio
and cellular phosphate
C12MBPa
Pretreatment
After 2 Weeks
Pretreatment
After 2 Weeks
10 1.02 + 0.26
10 1 67b 2 0.33
18 1 09 * 0.13
18 100 & 013
0.71 2 0.21 0.70 2 0.16
0.93c -+ 0 22 0.56d ? 0.15
0.62 ? 0.23 0.57 2 0.23
0.54 ? 0.24 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
257
and cellular phosphate
Table II. Aqueous organic phosphate
concentrations
in erythrocytes,
blood platelets, and leukocytes
of patients receiving oral bisphos-
phonates. Erythrocytesa,b CI,MBP
EHBP
No. of patients ATP (mmolil packed 2,3-DPG (mmolil
cells)
packed cells)
After 2 Weeks
Pretreatment
After 2 Weeks
Pretreatment
10 1.37 2 0.24
10 1.50 c 0 24
18 1.51 ” 0.32
145
4.85 f
457
4.14 -r- 047
3.92 * 1.32
1.05
-t 1.60
Leukocyte@
Piateletsa,b
No. of patients ATP (mmol/l cell water) No. of patients ADP (mmol/l cell water)
18 f 031
Pretreatment
After 2 Weeks
4 2.49 * 0.29 4 1.52 2 0.12
4 2 52 t 0.32 4 1 70 t 0.16
Pretreatment
After 2 Weeks
4 199 * 015 3 1.05 + 0 10
4 2 025 3 0.94 ? 0 10
219
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 hyperphosphatemic 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 intestrnal 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
Table Ill. Comparison
Pretreatment Posttreatment
of the distribution
of P, and chloride
dogs (Francis and Slough, 1984), in which hyperphosphatemia 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 contributed 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 Iuminal surface of the renal brush border membrane (Kessler and Fanestil, 1981) and suggest that an additional rate-limiting process is required before changes in tubular P, transport occur. For example, it has been suggested 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 biochemical 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
between erythrocytes
Plasma P, (mmolil plasma)”
T,,,/GFRa (mmolll glomerular filtrate (n = 4)
115 * 011 2 28b 2 0.24
1.06 2 0.16 2.40c ? 0 38
and plasma during treatment
Erythrocyteiplasma P, Ratioa (mmolil packed cells)
with intravenous
ErythrocyteiPlasma Ratioa (mmolil packed
EHBP. Chloride cells)
(mmolil plasma)
(mmol/l plasma)
0.61 + 0 08 0.39d * 0 09
0 60 lr 0.07 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
OLOff
Platelets
Erythrocytes
Plasma
OLOn
Off
Leukocytes
d- -
CJOn
Off
On
Off
On
Fig. 1. Comparison
of the responses of cellular P, to hyperphosphatemia induced by EHBP in erythrocytes, platelets, and leukocytes. 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 significantly less than that in plasma for all three cell types: erythrocytes 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 increased Pi retention. In an earlier study (Challa et al., 1985), we showed that in severely hypophosphatemic or hyperphosphatemic patients in an intensive care unit the erythrocyte P, concentrations were roughly proportional to those in plasma. These observations are consistent with passive distribution of P, across the cell membrane being the dominant factor determining the cellular P, concentration. In contrast, 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 proportionality 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, concentration 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). Therefore, 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-induced changes in the membrane potential, pH, or water content could alter the distribution of permeant anions, including P,, across the cell membrane (Hladky and Rink,
Bisphosphonates
and cellular phosphate
1977; but we showed that these factors were probably unimportant here, as the distribution of the passively transported 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, concentration at a lower value than that predicted from the Increase 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 reported in plasma (Miihlbauer and Fleisch, 1985). These findings are consistent with earlier work that suggested 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 measurements 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 oxygenation 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 membrane. This is consistent with the observation that, at normal extracellular P, concentrations (1 mmolil), erythrocytes 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 resonance 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 resrstance to change by cellular P, occurs in other forms of hypophosphatemia and hyperphosphatemia and whether it is a true regulatory mechanism or an indirect consequence 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