Acta med. scand. Vol. 197, pp. 357-360, 1975
ADULT HYPOPHOSPHATASIA Report of a Case with Determination of Inorganic Pyrophosphate in Plasma and Urine during High Phosphate Intake
Eli Ssrensen and Hans Flodgaard From Medical Department P , Division of Gastroenterology, Rigshospitalet, and Department of Biochemistry A, University of Copenhagen, Copenhagen, Denmark
Abstract. A case of adult hypophosphatasia under treatment with a high orthophosphate (PI)intake is described. The patient is a 53-year-old woman. Her symptoms have progressed for seven years, and it has been necessary to perform osteosynthesis of both cnrra. The diagnosis rests upon a characteristic clinical picture, low serum alkaline phosphatase activity, high urinary excretion of phosphoethanolamine, and an invariably elevated concentration of inorganic pyrophosphate (PPJ in plasma accompanied by a very high excretion of this compound in the urine. An improved technique allowed specific determinations of microquantities of PPI in biologic materials. The concentrations of PPI in plasma and urine remained unchanged when the patient’s intake of phosphorus was increased to 1.98 g/day. The PPl/Plratio in the urine was 10-20 before treatment. During treatment PI excretion increased, PPI excretion did not change, and the ratio decreased to around 7. The renal tubular transport of PPI probably was saturated, and therefore PP,, which was circulating in abnormally high concentrations in the patient’s fluids, could not be removed by loading with PI. Four months of treatment did not benefit the patient.
tional activity as inorganic pyrophosphatase (3), could remove the inhibition. Russell (13) hypothesized that the elevated PPi in the circulating fluids in hypophosphatasic patients could be a result of the deficient alkaline (pyro)phosphatase activity, while the impaired bone mineralization could be a result of the inhibitory effect of the abnormally high PPI.Consequently reduction of the PPIconcentration might be useful in treatment of patients with this disease. The urinary excretions of orthophosphate (PI) and of PPIare linked in normal subjects, probably by a common excretion mechanism in the kidney. Loading with PIincreases the excretion of both PI and PP1in the urine in normal subjects and in patients with urolithiasis (6). Treatment with PIhas been tried in patients with hypophosphatasia but the results reported are incomplete ( 2 , 8 , 14). The methods used so far to measure PP,in biological fluids have been based upon tedious and nonspecific chromatographic separations of PPIfrom other phosphate compounds, and have given variable results for plasma concentrations of PPI(13,
Hypophosphatasia is an inborn error of metabolism, characterized by skeletal and dental deformities, subnormal activity of serum alkaline phosphatase, and excessive excretion of phos- 14). About 200 cases of hypophosphatasia, most of phoethanolamine (1). Recently, Russell (13), and Russell et al. (14) reported invariably elevated them children, have been described. Only 14 adult concentrations of inorganic pyrophosphate (PPJin cases have been recognized ( I , 9, 10, 11). We deplasma and urine from patients suffering from this scribe a case of adult hypophosphatasia with a disease. These findings are important because progressive course. Before and during treatment Heisch and Bisaz (5) have found that PPIeffectively with high phosphate intake the plasma and urinary inhibits collagen-induced calcium phosphate pre- concentrations of PPIhave been determined by a cipitation in vitro. They demonstrated that alkaline sensitive and specific isotope derivative method for phosphatase, which is known to possess an addi- determination of PP,in biological material (7). Acta med. scand. 197
358
E. Ssrensen and H . Flodgaard
Table I . Plasma concentrations and urinary excretion of inorganic pyrophosphate (PPJ before and during treatment with a high phosphate intake
Before treatment 3 1 6.26
1.22
2.450 1.675
82.19 213.30
During treatment with a high phosphate intake 2 5.78 1.53 1.950 180.10 8 4.80 1.59 2.175 159.50 15 4.86 1.04 1.800 194.30 22 6.27 1.62 2.150 112.50 34 6.49 1.19 69 8.19 1.25 89 8.46 1.22 2.550 163.34 125 9.21 1.35 2.460 322.08
7.89 9.94
201.4 357.3
19.3 16.7
10.4 21.5
27.43 18.26 23.45 17.71
351.2 346.9 349.7 241.9
53.5 39.7 42.21 38.1
6.6 8.7 8.3 6.4
21.91 48.18
416.52 792.32
55.87 118.52
7.5 6.7
METHODS Blood was drawn by venipuncture as atraumatically as possible into 5 ml heparinized plastic vessels surrounded by ice. It is important that no coagulation occurs because release of PPI from platelets is well known (15). The plasma was immediately separated by centrifugation in the cold and 5 volumes of ice-cold perchloric acid were added. After standing for 10 min in the cold the precipitate was removed by centrifugation and the supernatant neutralized with ice-cold KOH. This procedure has been found to give 100 % recovery of PP1. The urine was treated with perchloric acid in a similar way in order to remove any inorganic pyrophosphatase activity. Such treatment of urine gave 100% recovery of PPI. The analytical method for PPI determination has been described elsewhere (7). The PI was measured by the method of Wahler and Wollenberger (16).
CASEREPORT Previous history The patient is a 53-year-old married woman. There is no family history of bone diseases, but two brothers died in infancy from unknown causes. Her mother has old-age diabetes. One year old the patient was admitted to a sanatorium for one year because of “rickets”. She did not walk until two years old. She lost her permanent teeth because of “fragile enamel” at the age of 15. She was pregnant only once, but had a spontaneous abortion. At the age of 29 and 39 cysts were removed from her ovaries. After menopause at 45, she had oestrogenl androgen (Femovirin”, 1 ml) idections every month because of climacteric symptoms. Since 40 she has often suffered from headache for which she has been taking up to 4 g aspirin a day. Acta med. scand. 197
Actual bone and joint history In 1960 the patient had a Colles fracture of the left wrist after adequate trauma and with prompt healing. In 1966 she developed pains in her right foot. X-ray was normal, but two years later several fractures of the metatarsal bones were seen. With the exception of one year-1970-when the patient was able to resume her work in a factory, she gradually developed incapacitating pains in her feet, knees, hips, low back and wrist, and successive fractures in the calcaneus, processus styloideus of the ulna and radius, both tibiae and fibulae as well as pseudofractures of both femora. A general progressive halisteresis was observed and radiological and clinical signs of arthroses in both lower extremities, as well as calcification of the spinal ligaments. Diagnosis Blood examinations were not carried out until 1970, when a very low alkaline phosphatase activity was found. Later in the same year a search for hyperparathyroidism was made with negative result. In 1973 hypophosphatasia was suspected because of the invariably subnormal alkaline phosphatase activities and the diagnosis was confirmed by the finding of a 24-hour urinary excretion of 244 pmoles phosphoethanolamine, a value far above normal (12). Traces of the same compound were found in plasma. A bone biopsy taken in 1973 contained considerable amounts of uncalcified osteoid. Alkaline phosphatase activity was normal in biopsy from the small intestine. Histochemical examination of granulocytes for alkaline phosphatase activity gave the very low score of 4 (NR 4CL100). Other laboratory investigations The patient’s height was 155 cm, weight 55 kg, BP 140190. Total blood count was normal, ESR 7 mm/h. Alkaline phosphatase in serum was 3-10 Ull (NR 13-38, modified
Adult hypophosphatasia Bessey-Lowry); isoenzyme determinations were not possible, the total activity being too low. Acid phosphatase was 7.1 U/l (2.7-10.5). Alanine aminotransferase, a-amylase, bilirubin and prothrombin were normal. Serum electrophoresis normal. In plasma the following cocentrations were found: calcium 2.53 mM, phosphate 1.56 mM, sodium 142 mM, potassium 3.9 mM, standard bicarbonate 24.2 mM, and magnesium 0.83 mM. Serum creatinine was 0.09 mM, carbamide 7.8 mM, creatinine clearance 0.94 ml/sec (NR 1.6-2.0). A 24-hour urine contained 4.6 mmoles calcium and 17 mmoles phosphate. There was no albumin, blood or glucose in the urine, and microscopy was normal. Traces of blood were found in her feces; 9 mmoles lipid were excreted in a 24-hour feces sample. Radiologically, her chest, cranium and stomach appeared normal. Intravenous pyelograms were slightly contracted. ECGs were normal. Serum thyroxine was 95 nM. A calcium balance study showed normal results and measurements of mineral contents in bone showed low normal values.
359
whereas the plasma values are about half the magnitude reported by Russell et al. (14). The plasma PI concentration increased slightly during the high phosphate intake, while the urinary excretion augmented to about three times the pretreatment value (Table I). The plasma PPI concentration and the urinary excretion did not change. Consequently the PPl/Pl ratio fell to about 7, which is still far above the ratio of approximately 0.2 found in normal subjects (4).
DISCUSSION
More than 10 cases of adult hypophosphatasia have now been described (1, 9, 10, 11). In most of them the disease has been misinterpreted in early childhood as rickets. There is no sex preference, though the tendency of postmenopausal women to develop osteoporosis may provoke some latent cases. Typically, there are symmetric fractures and pseudofractures of the lower extremities. Most of the reOrthopedic treatment ported adult cases had symmetrical pseudofractures At first, in 1967, when no bone or joint lesions had been found, the patient's pains were interpreted as muscular of the femoral diaphyses. Progressive halisteresis and treated with physical therapy. Later, when multiple has been noted in several cases as well as calcifractures were found in her right foot, this was im- fication of the spinal ligaments, and chondromobilized with good result. In 1972 the patient had pains calcinosis (1 1). In many respects, our patient had in her left knee and crus for 4 months before a fracture was diagnosed. Even 4 months of immobilization did not result the characteristic syndrome. Since Fleisch and Bisaz ( 5 ) showed that PPi conin healing. In March 1973, therefore, an osteosynthesis of the left tibia was performed. Increasing consolidation re- centrations in the range 10-5-10-7 M effectively insulted, but one year later the fracture line was still visible. hibited collagen-induced calcium phosphate preA somewhat similar course was observed for the right cipitation in vitro, it is tempting to speculate that tibia. The fracture line was first seen in Nov. 1973 but as the abnormally high PP, concentrations in the the patient hardly ever stood on her legs, but moved around using crutches or in a wheelchair, no further biological fluids in hypophosphatasic patients may treatment was found necessary. However the fracture line prevent normal calcification. Russell (13) suggested became more and more distinct and an osteosynthesis of that a high phosphate intake might lead to imthe right tibia was performed in May 1974. provement in this disease. In a later paper (14), however, he stated (without documentation) that Phosphate treatment such treatment did not change PP1 concentrations The patient has never received cortisone, calcium or in plasma and urine. This is in agreement with our vitamin D treatment. results, and may explain why a beneficial effect in A preparation of disodium hydrogen phosphate, 300 mg, and potassium dihydrogen phosphate, 200 mg, was used. our patient has been lacking so far. It has proved The patient received 12 g of this compound a day, as 8 impossible to increase the PP1 excretion by loading tablets three times a day, which corresponds to an inwith Pi. In normal subjects an increased Pi excrecreased phosphorus intake of I .98 glday . tion is accompanied by an increased PP, excretion, Before onset of the high phosphate intake the patient was admitted to hospital and kept on a standard diet so that the PP,/Pi ratio remains constant (4). The containing 1260 mg phosphorus per day in order to renal tubular transport of PP, in our patient may establish a pretreatment level of plasma PP,. have been saturated and this would explain why The plasma concentration and the urinary excretion of PP, were elevated before as well as during treatment (Ta- PP1, circulating in an abnormally high concentrable I). The normal concentration of PPI in plasma is tion, cannot be removed from the body. In the cases 1.92k0.092 pM (S.E.M., n=23), determined by the reported by Bongiovanni et al. (2) and Guibaud and isotope derivative method. The excretion of PP, in urine Larbre (8), the treated children showed significant from volunteers of the same age as the patient has been found to be in the region of 30 pmoIes/24 hours. This is in clinical and radiological improvement. Bongiovanni good agreement with the findings of Fleisch and Bisaz (4) et al. found a rise in urinary pyrophosphate, which Acta med. scand. 197
360
E. S ~ e n s e nand H . Flodgaard
has not been confirmed by Guibaud and Larbre or the present work. We did not find any improvement clinically or radiologically during the treatment, which may be due to our patient’s age. The isotope derivative method used in the present study for measuring PPI is specific and very sensitive (lo-$ M can be measured in a sample volume of 50 ~1 with an S.D. less than 5%). More correct concentrations of PP1in plasma from normal subjects (1.92f0.092 pM) and from a hypophosphatasic patient have now been established. The higher values for plasma PP1both in normals and in hypophosphatasia reported by Russell et al. (14) must be due to a systemic error. Their conclusion, that PPIis invariably elevated in plasma and urine in this disease, is confirmed by the present work. Sitcox and McCarty (15) recently reported a PPI concentration in plasma of 1Af0.06 pM (S.E.M.) from 94 normal subjects. The essential step in their procedure is based upon a specific enzyme, and is in very good agreement with our results.
REFERENCE§ I . Bartter, F. C.: Hypophosphatasia. In: The metabolic basis of inherited disease (ed. Stanbury, Wyngaarden & Fredrickson), 3rd edition, p. 1295. McGraw Hill, New York, Toronto, Sydney and London 1972. 2. Bongiovanni, A. M., Album, M. M., Root, A. W., Hope, J. W., Marino, J. & Spencer, D. M.: Studies in hypophosphatasia and response to high phosphate intake. Arner. J. Med. Sci. 255: 163, 1968. 3. Femley, H. N. &Walker, P. G.: Studies on alkaline phosphatases: Inhibition by phosphate derivatives and the substrate specificity. Biochem. J. 104: 1011, 1967. 4. Fleisch, H. & Bisaz, S.: Die Pyrophosphataus-
Acta med. scund. 197
scheidung im Ham beim gesunden Menschen. Helv. physiol. Acta 21: 88, 1963. 5. Isolation from urine of pyrophosphate, a calcification inhibitor. Amer. J. Physiol. 203: 671, 1962. 6. Fleisch, H., Bisaz, S. & Care, A. D.: Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet 1: 1065,
-
1%. 7. Flodgaard,
H. & Fleron, P.: Thermodynamic parameters for the hydrolysis of inorganic pyrophosphate at pH 7.4 as a function of Mg*+, K+, and ionic strength determined from equilibrium studies of the reaction. J. biol. Chem. 249: 3465, 1974. 8. Guibaud, P. & Larbre, F.: A propos d’un cas d’hypophosphatasie ti forme benigne traitee par le phosphore. Pkdiatrie 25: 3 19, 1970. 9. Hosenfeld, D. & Hosenfeld, A.: Qualitative und quantitative Untersuchungen der Isoenzyme der alkalischen Serumphosphatase bei der Hypophosphatasie. Klin. Padiat. 185:437, 1973. 10. Jardon, 0. M.,Burney, D. W. & Fink, R. L.: Hypophosphatasia in an adult. J. Bone Jt Surg. 52-A: 1477, 1970. 11. O’Duffy, J. D.: Hypophosphatasia associated with
calcium pyrophosphate dihydrate deposits in cartilage. Report of a case. Arthr. and Rheum. 13: 381, 1970. 12. Rasmussen, K.: Phosphorylethanolamine and hypophosphatasia. Thesis, Aarhus 1968. 13. Russell, R. G. G.: Excretion of inorganic pyrophosphate in hypophosphatasia. Lancet 2: 461, 1%5. 14. Russell, R. G. G., Bisaz, S., Donath, A., Morgan, D. B. & Fleisch, H.:Inorganic pyrophosphate in plasma
in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone. J. clin. Invest. 50: 961, 1971. 15. Silcox, D. C. & McCarty, D. J.: Measurement of inorganic pyrophosphate in biological fluids. J. clin. Invest. 52: 1863, 1973. 16. Wahler, B. E. & Wollenberger, A.: Zur Bestimmung des Orthophosphats neben saure-rnolybdat-labilen Phosphorsaureverbindungen. Biochem. Z. 329: 508, 1958.
ADULT HYPOPHOSPHATASIA Report of a Case with Determination of Inorganic Pyrophosphate in Plasma and Urine during High Phosphate Intake
Eli Ssrensen and Hans Flodgaard From Medical Department P , Division of Gastroenterology, Rigshospitalet, and Department of Biochemistry A, University of Copenhagen, Copenhagen, Denmark
Abstract. A case of adult hypophosphatasia under treatment with a high orthophosphate (PI)intake is described. The patient is a 53-year-old woman. Her symptoms have progressed for seven years, and it has been necessary to perform osteosynthesis of both cnrra. The diagnosis rests upon a characteristic clinical picture, low serum alkaline phosphatase activity, high urinary excretion of phosphoethanolamine, and an invariably elevated concentration of inorganic pyrophosphate (PPJ in plasma accompanied by a very high excretion of this compound in the urine. An improved technique allowed specific determinations of microquantities of PPI in biologic materials. The concentrations of PPI in plasma and urine remained unchanged when the patient’s intake of phosphorus was increased to 1.98 g/day. The PPl/Plratio in the urine was 10-20 before treatment. During treatment PI excretion increased, PPI excretion did not change, and the ratio decreased to around 7. The renal tubular transport of PPI probably was saturated, and therefore PP,, which was circulating in abnormally high concentrations in the patient’s fluids, could not be removed by loading with PI. Four months of treatment did not benefit the patient.
tional activity as inorganic pyrophosphatase (3), could remove the inhibition. Russell (13) hypothesized that the elevated PPi in the circulating fluids in hypophosphatasic patients could be a result of the deficient alkaline (pyro)phosphatase activity, while the impaired bone mineralization could be a result of the inhibitory effect of the abnormally high PPI.Consequently reduction of the PPIconcentration might be useful in treatment of patients with this disease. The urinary excretions of orthophosphate (PI) and of PPIare linked in normal subjects, probably by a common excretion mechanism in the kidney. Loading with PIincreases the excretion of both PI and PP1in the urine in normal subjects and in patients with urolithiasis (6). Treatment with PIhas been tried in patients with hypophosphatasia but the results reported are incomplete ( 2 , 8 , 14). The methods used so far to measure PP,in biological fluids have been based upon tedious and nonspecific chromatographic separations of PPIfrom other phosphate compounds, and have given variable results for plasma concentrations of PPI(13,
Hypophosphatasia is an inborn error of metabolism, characterized by skeletal and dental deformities, subnormal activity of serum alkaline phosphatase, and excessive excretion of phos- 14). About 200 cases of hypophosphatasia, most of phoethanolamine (1). Recently, Russell (13), and Russell et al. (14) reported invariably elevated them children, have been described. Only 14 adult concentrations of inorganic pyrophosphate (PPJin cases have been recognized ( I , 9, 10, 11). We deplasma and urine from patients suffering from this scribe a case of adult hypophosphatasia with a disease. These findings are important because progressive course. Before and during treatment Heisch and Bisaz (5) have found that PPIeffectively with high phosphate intake the plasma and urinary inhibits collagen-induced calcium phosphate pre- concentrations of PPIhave been determined by a cipitation in vitro. They demonstrated that alkaline sensitive and specific isotope derivative method for phosphatase, which is known to possess an addi- determination of PP,in biological material (7). Acta med. scand. 197
358
E. Ssrensen and H . Flodgaard
Table I . Plasma concentrations and urinary excretion of inorganic pyrophosphate (PPJ before and during treatment with a high phosphate intake
Before treatment 3 1 6.26
1.22
2.450 1.675
82.19 213.30
During treatment with a high phosphate intake 2 5.78 1.53 1.950 180.10 8 4.80 1.59 2.175 159.50 15 4.86 1.04 1.800 194.30 22 6.27 1.62 2.150 112.50 34 6.49 1.19 69 8.19 1.25 89 8.46 1.22 2.550 163.34 125 9.21 1.35 2.460 322.08
7.89 9.94
201.4 357.3
19.3 16.7
10.4 21.5
27.43 18.26 23.45 17.71
351.2 346.9 349.7 241.9
53.5 39.7 42.21 38.1
6.6 8.7 8.3 6.4
21.91 48.18
416.52 792.32
55.87 118.52
7.5 6.7
METHODS Blood was drawn by venipuncture as atraumatically as possible into 5 ml heparinized plastic vessels surrounded by ice. It is important that no coagulation occurs because release of PPI from platelets is well known (15). The plasma was immediately separated by centrifugation in the cold and 5 volumes of ice-cold perchloric acid were added. After standing for 10 min in the cold the precipitate was removed by centrifugation and the supernatant neutralized with ice-cold KOH. This procedure has been found to give 100 % recovery of PP1. The urine was treated with perchloric acid in a similar way in order to remove any inorganic pyrophosphatase activity. Such treatment of urine gave 100% recovery of PPI. The analytical method for PPI determination has been described elsewhere (7). The PI was measured by the method of Wahler and Wollenberger (16).
CASEREPORT Previous history The patient is a 53-year-old married woman. There is no family history of bone diseases, but two brothers died in infancy from unknown causes. Her mother has old-age diabetes. One year old the patient was admitted to a sanatorium for one year because of “rickets”. She did not walk until two years old. She lost her permanent teeth because of “fragile enamel” at the age of 15. She was pregnant only once, but had a spontaneous abortion. At the age of 29 and 39 cysts were removed from her ovaries. After menopause at 45, she had oestrogenl androgen (Femovirin”, 1 ml) idections every month because of climacteric symptoms. Since 40 she has often suffered from headache for which she has been taking up to 4 g aspirin a day. Acta med. scand. 197
Actual bone and joint history In 1960 the patient had a Colles fracture of the left wrist after adequate trauma and with prompt healing. In 1966 she developed pains in her right foot. X-ray was normal, but two years later several fractures of the metatarsal bones were seen. With the exception of one year-1970-when the patient was able to resume her work in a factory, she gradually developed incapacitating pains in her feet, knees, hips, low back and wrist, and successive fractures in the calcaneus, processus styloideus of the ulna and radius, both tibiae and fibulae as well as pseudofractures of both femora. A general progressive halisteresis was observed and radiological and clinical signs of arthroses in both lower extremities, as well as calcification of the spinal ligaments. Diagnosis Blood examinations were not carried out until 1970, when a very low alkaline phosphatase activity was found. Later in the same year a search for hyperparathyroidism was made with negative result. In 1973 hypophosphatasia was suspected because of the invariably subnormal alkaline phosphatase activities and the diagnosis was confirmed by the finding of a 24-hour urinary excretion of 244 pmoles phosphoethanolamine, a value far above normal (12). Traces of the same compound were found in plasma. A bone biopsy taken in 1973 contained considerable amounts of uncalcified osteoid. Alkaline phosphatase activity was normal in biopsy from the small intestine. Histochemical examination of granulocytes for alkaline phosphatase activity gave the very low score of 4 (NR 4CL100). Other laboratory investigations The patient’s height was 155 cm, weight 55 kg, BP 140190. Total blood count was normal, ESR 7 mm/h. Alkaline phosphatase in serum was 3-10 Ull (NR 13-38, modified
Adult hypophosphatasia Bessey-Lowry); isoenzyme determinations were not possible, the total activity being too low. Acid phosphatase was 7.1 U/l (2.7-10.5). Alanine aminotransferase, a-amylase, bilirubin and prothrombin were normal. Serum electrophoresis normal. In plasma the following cocentrations were found: calcium 2.53 mM, phosphate 1.56 mM, sodium 142 mM, potassium 3.9 mM, standard bicarbonate 24.2 mM, and magnesium 0.83 mM. Serum creatinine was 0.09 mM, carbamide 7.8 mM, creatinine clearance 0.94 ml/sec (NR 1.6-2.0). A 24-hour urine contained 4.6 mmoles calcium and 17 mmoles phosphate. There was no albumin, blood or glucose in the urine, and microscopy was normal. Traces of blood were found in her feces; 9 mmoles lipid were excreted in a 24-hour feces sample. Radiologically, her chest, cranium and stomach appeared normal. Intravenous pyelograms were slightly contracted. ECGs were normal. Serum thyroxine was 95 nM. A calcium balance study showed normal results and measurements of mineral contents in bone showed low normal values.
359
whereas the plasma values are about half the magnitude reported by Russell et al. (14). The plasma PI concentration increased slightly during the high phosphate intake, while the urinary excretion augmented to about three times the pretreatment value (Table I). The plasma PPI concentration and the urinary excretion did not change. Consequently the PPl/Pl ratio fell to about 7, which is still far above the ratio of approximately 0.2 found in normal subjects (4).
DISCUSSION
More than 10 cases of adult hypophosphatasia have now been described (1, 9, 10, 11). In most of them the disease has been misinterpreted in early childhood as rickets. There is no sex preference, though the tendency of postmenopausal women to develop osteoporosis may provoke some latent cases. Typically, there are symmetric fractures and pseudofractures of the lower extremities. Most of the reOrthopedic treatment ported adult cases had symmetrical pseudofractures At first, in 1967, when no bone or joint lesions had been found, the patient's pains were interpreted as muscular of the femoral diaphyses. Progressive halisteresis and treated with physical therapy. Later, when multiple has been noted in several cases as well as calcifractures were found in her right foot, this was im- fication of the spinal ligaments, and chondromobilized with good result. In 1972 the patient had pains calcinosis (1 1). In many respects, our patient had in her left knee and crus for 4 months before a fracture was diagnosed. Even 4 months of immobilization did not result the characteristic syndrome. Since Fleisch and Bisaz ( 5 ) showed that PPi conin healing. In March 1973, therefore, an osteosynthesis of the left tibia was performed. Increasing consolidation re- centrations in the range 10-5-10-7 M effectively insulted, but one year later the fracture line was still visible. hibited collagen-induced calcium phosphate preA somewhat similar course was observed for the right cipitation in vitro, it is tempting to speculate that tibia. The fracture line was first seen in Nov. 1973 but as the abnormally high PP, concentrations in the the patient hardly ever stood on her legs, but moved around using crutches or in a wheelchair, no further biological fluids in hypophosphatasic patients may treatment was found necessary. However the fracture line prevent normal calcification. Russell (13) suggested became more and more distinct and an osteosynthesis of that a high phosphate intake might lead to imthe right tibia was performed in May 1974. provement in this disease. In a later paper (14), however, he stated (without documentation) that Phosphate treatment such treatment did not change PP1 concentrations The patient has never received cortisone, calcium or in plasma and urine. This is in agreement with our vitamin D treatment. results, and may explain why a beneficial effect in A preparation of disodium hydrogen phosphate, 300 mg, and potassium dihydrogen phosphate, 200 mg, was used. our patient has been lacking so far. It has proved The patient received 12 g of this compound a day, as 8 impossible to increase the PP1 excretion by loading tablets three times a day, which corresponds to an inwith Pi. In normal subjects an increased Pi excrecreased phosphorus intake of I .98 glday . tion is accompanied by an increased PP, excretion, Before onset of the high phosphate intake the patient was admitted to hospital and kept on a standard diet so that the PP,/Pi ratio remains constant (4). The containing 1260 mg phosphorus per day in order to renal tubular transport of PP, in our patient may establish a pretreatment level of plasma PP,. have been saturated and this would explain why The plasma concentration and the urinary excretion of PP, were elevated before as well as during treatment (Ta- PP1, circulating in an abnormally high concentrable I). The normal concentration of PPI in plasma is tion, cannot be removed from the body. In the cases 1.92k0.092 pM (S.E.M., n=23), determined by the reported by Bongiovanni et al. (2) and Guibaud and isotope derivative method. The excretion of PP, in urine Larbre (8), the treated children showed significant from volunteers of the same age as the patient has been found to be in the region of 30 pmoIes/24 hours. This is in clinical and radiological improvement. Bongiovanni good agreement with the findings of Fleisch and Bisaz (4) et al. found a rise in urinary pyrophosphate, which Acta med. scand. 197
360
E. S ~ e n s e nand H . Flodgaard
has not been confirmed by Guibaud and Larbre or the present work. We did not find any improvement clinically or radiologically during the treatment, which may be due to our patient’s age. The isotope derivative method used in the present study for measuring PPI is specific and very sensitive (lo-$ M can be measured in a sample volume of 50 ~1 with an S.D. less than 5%). More correct concentrations of PP1in plasma from normal subjects (1.92f0.092 pM) and from a hypophosphatasic patient have now been established. The higher values for plasma PP1both in normals and in hypophosphatasia reported by Russell et al. (14) must be due to a systemic error. Their conclusion, that PPIis invariably elevated in plasma and urine in this disease, is confirmed by the present work. Sitcox and McCarty (15) recently reported a PPI concentration in plasma of 1Af0.06 pM (S.E.M.) from 94 normal subjects. The essential step in their procedure is based upon a specific enzyme, and is in very good agreement with our results.
REFERENCE§ I . Bartter, F. C.: Hypophosphatasia. In: The metabolic basis of inherited disease (ed. Stanbury, Wyngaarden & Fredrickson), 3rd edition, p. 1295. McGraw Hill, New York, Toronto, Sydney and London 1972. 2. Bongiovanni, A. M., Album, M. M., Root, A. W., Hope, J. W., Marino, J. & Spencer, D. M.: Studies in hypophosphatasia and response to high phosphate intake. Arner. J. Med. Sci. 255: 163, 1968. 3. Femley, H. N. &Walker, P. G.: Studies on alkaline phosphatases: Inhibition by phosphate derivatives and the substrate specificity. Biochem. J. 104: 1011, 1967. 4. Fleisch, H. & Bisaz, S.: Die Pyrophosphataus-
Acta med. scund. 197
scheidung im Ham beim gesunden Menschen. Helv. physiol. Acta 21: 88, 1963. 5. Isolation from urine of pyrophosphate, a calcification inhibitor. Amer. J. Physiol. 203: 671, 1962. 6. Fleisch, H., Bisaz, S. & Care, A. D.: Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet 1: 1065,
-
1%. 7. Flodgaard,
H. & Fleron, P.: Thermodynamic parameters for the hydrolysis of inorganic pyrophosphate at pH 7.4 as a function of Mg*+, K+, and ionic strength determined from equilibrium studies of the reaction. J. biol. Chem. 249: 3465, 1974. 8. Guibaud, P. & Larbre, F.: A propos d’un cas d’hypophosphatasie ti forme benigne traitee par le phosphore. Pkdiatrie 25: 3 19, 1970. 9. Hosenfeld, D. & Hosenfeld, A.: Qualitative und quantitative Untersuchungen der Isoenzyme der alkalischen Serumphosphatase bei der Hypophosphatasie. Klin. Padiat. 185:437, 1973. 10. Jardon, 0. M.,Burney, D. W. & Fink, R. L.: Hypophosphatasia in an adult. J. Bone Jt Surg. 52-A: 1477, 1970. 11. O’Duffy, J. D.: Hypophosphatasia associated with
calcium pyrophosphate dihydrate deposits in cartilage. Report of a case. Arthr. and Rheum. 13: 381, 1970. 12. Rasmussen, K.: Phosphorylethanolamine and hypophosphatasia. Thesis, Aarhus 1968. 13. Russell, R. G. G.: Excretion of inorganic pyrophosphate in hypophosphatasia. Lancet 2: 461, 1%5. 14. Russell, R. G. G., Bisaz, S., Donath, A., Morgan, D. B. & Fleisch, H.:Inorganic pyrophosphate in plasma
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