Original Article
Ann Clin Biochem 1992; 29: 529-534
Pyrophosphate in synovial fluid and urine and its relationship to urinary risk factors for stone disease N B Roberts, J Dutton, T HelIiweUl, P J N Rothwell- and J P KavanaghFrom the Departments of Chemical Pathology and [Pathology, Royal Liverpool Hospital, Liverpool, £"7 8XW and 2Department of Urology, University Hospital of South Manchester, Manchester M20 8LR, UK
Inorganic pyrophosphate (PP j ) measurement in urine and synovial fluid has been established using the PPj-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 JLmollL, easy to perform and gives results comparable to more complex methods. Daily urinary output of PP j was independently related to both age (P= 0.(014) and sex (P=0·OOO2). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P=O·012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PP j excretion with .urine volume (P = 0·004) and with phosphate excretion (P=0·008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPj. PPj excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PP j concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPj or urate crystals.
SUMMARY.
Additional key phrases: urine phosphate; urine oxalate; hypophosphatasia; arthritis
Inorganic pyrophosphate (PPj) is produced by metabolic pathways involving phosphoribonucleotides. The cumulative daily turnover in humans maybe in the order of kilograms and plasma PP j has a half-life of about 2 min.' Only relatively small amounts of free PP j are actually measurable in plasrna.? urine;' in the mineral of bone" and teeth.' The measurement of an increased excretion of pyrophosphate in urine is the basis for confirmation of the diagnosis of the rare inherited disorder hypophosphatasia." In synovial fluid PPj has been measured in investigations of chondrocalcinosis or pyrophosphate arthropathy (pseudo gout) and other joint disorders.V A low urinary output of PP j could contribute to an increased risk of stone formation as it is well established that PP j can act as an inhibitor of crystallization of various renal stone forming components (of calcium oxalate.P-" brushite and apatite!' and struvite"). Correspondence: Dr N B Roberts.
Russell and Hodgkinson" measured PPj in the urine from stone formers using small ion-exchange columns, but the procedure was laborious and impractical for routine purposes. PPj has also been measured using the enzyme uri dine 5 1_ diphosphoglucose pyrophosphorylase, either with radiolabelled substrates'45: stone stone stone
·
Y .:
..
... :. ....:..........
....:
...
None
Urate
~
PP j
Crystal cantent of synavial fluid FIGURE 3. Pyrophosphate concentrations in synovial fluid from patients with painful, swollen knees; classified by the crystal content of the synovial fluid.
Downloaded from acb.sagepub.com by guest on June 4, 2016
Pyrophosphate in synovial fluid and urine DISCUSSION The analytical procedure described by the manufacturer was modified to enable the simultaneous analysis of up to 16 tests in 1.0 mL volumes. We found the assay was simple to use and had acceptable precision and good recovery for the analysis of PP j in urine or synovial fluid. The arithmetic mean of urinary PP j excretion in healthy controls (33' 5 fLmol/day) compares well with previously reported values, 36' 5 13 and 39' 3 fLmol/day,21 obtained by two different methods. While this is reassuring, it is of limited significance as we observed associations between PP j and age and sex. In addition, the distribution was not symmetrical and the geometric mean (23' 2 fLmol/day) is a better estimate of the central tendency of the spread of results. Schwille et al. ,IS using a specific radioisotopic technique, also note skewed distributions and the importance of age and sex matching. The ranges of PP j in their controls « = 40 years) were 15-98 (men) and 4-46 (women) fLmol/day; our ranges « = 45 years) were very similar at 15-108 (men) and 6-40 (women) fLmol/day. An alternative enzymatic sequence has been used to adapt the method to a centrifugal analyser. 21 However, we have observed that centrifugation of urine can cause a decrease in the absorbance of some urines due to changes in turbidity and not to any chemical reaction. In these cases PP j could be overestimated. The relatively viscous nature of synovial fluid makes it impossible to adapt the assay on this type of sample to a centrifugal analyser, or in fact to any automated system with a fine needle probe. We have therefore adopted the manual procedure for measurement of PP j in urine and synovial fluid. No chemical interferences have been observed from either of these matrices using these enzyme procedures. 7 ,21 There are theoretical and experimental grounds for supposing deficient urinary PP j to be a risk factor for increased urolithogenesisv " but in practice this has not been a common finding. 13,22 This may be due in part to poor age matching of control samples, which if drawn from laboratory personnel would probably have a lower age range and a higher proportion of women than a typical group of stone formers. Both factors would tend to diminish differences between cases and controls, Schwille et al. ,IS who took age and sex into account, found lower PP j urinary output in older stone forming men and younger stone forming women, while we observed this in
533
younger male stone patients. In both studies the general trend is for stone formers to have lower urinary PP j than age and sex matched controls, while the degree of significance attained may be limited by relatively small group numbers. PP j deficiency may be a contributory factor to the aetiology of stone disease, particularly in young women, amongst whom the lowest levels of PP j excretion were found. Calcium oxalate stone formers amongst this group may be most appropriate for supplementation therapy with orthophosphate, which has been shown to increase PPi excretion.P In patients with renal stone disease the 24 h excretion of PP j was correlated only to volume and phosphate excretion (significantly and independently) and not to other important risk factors for stone disease. In practice, the urinary concentration of PP j will be the determining factor in its crystallization inhibitory activity and this decreases with increasing volumes despite the positive association between PP j amount and volume. The usual advice to stone formers to increase their fluid intake may therefore be counter productive if low urinary PP j was a contributory cause of their stone forming diathesis. This may, however, be more than overcome by the decrease in urinary supersaturation. The demonstration of increased urinary PP j excretion has proved most useful in the confirmatory diagnosis of hypophosphatasia. Increased urine PPi outputs were associated with active disease, as reported by Russell. 6 Interestingly, one of the patients had a daughter who did not have active bone disease but had a low plasma alkaline phosphatase (20 fL/L) and a normal PP j excretion (34 fLmol/day). Thus, a low plasma alkaline phosphatase activity is not necessarily associated with active metabolic bone disease in hypophosphatasia and the observation of an elevated urine PPi output may help to establish the onset of bony pathological processes in patients with this disease. Measurement of PP j concentrations in synovial fluid was not helpful in distinguishing pyrophosphate arthropathy from other causes of joint inflammation. Although PP j concentrations were elevated (> 10 fLmol/U) in all four cases associated with PP j crystals, raised levels were also found in 700/0 of samples with urate or no crystal inclusions. The increase in PP j may therefore simply be as a result of increased cellular damage and not related to any particular pathological event.
Downloaded from acb.sagepub.com by guest on June 4, 2016
534
Roberts et al.
The simple and reliable procedure outlined for the measurement of PP j in urine and synovial fluid, without the use of radioisotopes, will ease the routine monitoring and enhance investigations into the role of PP j in the various diseases in which it has been implicated. Acknowledgement We would like to thank Julie A Morris (Department of Statistics, Withington Hospital, Manchester, UK) for advice and for performing the statistical analyses. REFERENCES 1 Dieppe P, Calvert P. Crystals and Joint Disease. London: Chapman and Hall, 1983 2 Fleisch H, Bisaz S. Mechanism of calcification: Inhibitory role of pyrophosphate. Nature 1962; 195: 911
3 Fleisch H, Bisaz S. Isolation from urine of pyrophosphate, a calcification inhibitor. Amer J Physiol 1962; 203: 671-5 4 Perkins HR, Walker P G. The occurrence of pyrophosphate in bone. J Bone Joint Surg 1958; 4OB: 333-6 5 Bisaz S, Russell RGG, Fleisch H. Isolation of inorganic pyrophosphate from bovine and human teeth. Arch Oral Bioi 1968; 13: 683-96 6 Russell RGG. Excretion of pyrophosphate in hypophosphatasia. Lancet 1965; ii: 461-4 7 Altman RD, Muniz OE, Pita IC, Howell DS. Articular chondrocalcinosis microanalysis of pyrophosphate (PPi) in synovial fluid and plasma. Arth Rheum 1973; 16: 171-8 8 Pattrick, M, Hamilton E, Hornby I, Doherty M. Synovial fluid pyrophosphate and nucleoside triphosphate pyrophosphatase: comparison between normal and diseased joints and between inflamed and non-inflamed joints. Ann Rheum Dis 1991; 50: 214-18 9 Robertson WG, Peacock M, Nordin BEC. Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1973; 43: 31-7 10 Grases F, Genesterm G, Conte A, March P, CostaBauza A. Inhibitory effect of pyrophosphate, citrate, magnesium on chondroitin sulphate in calcium oxalate urolithiasis. Br J Urol 1989; 64: 235-7
11 Fleisch H. Inhibitors and promotors of stone formation. Kidney Int 1978; 13: 361-71 12 Mclean RIC, Downey I, Clapham L, Wilson IWL, Nickel IC. Pyrophosphate inhibitors of Proteus mirabilis induced struvite crystalization in vitro. Clin Chim Acta 1991; 20: 107-18 13 Russell ROO, Hodgkinson A. The urinary excretion of inorganic pyrophosphate by normal subjects and patients with renal calculus. Clin Sci 1966;31: 51-62 14 Cheung CP, Suhadolnik RI. Analysis of inorganic pyrophosphate at the picomole level. Anal Biochem 1977; 83: 61-3 15 Schwille PO, Rimenapf G, Wo1fel G, Kohler R. Urinary pyrophosphate in patients with recurrent calcium urolithiasis and in healthy controls: A Reevaluation. J Urol 1988; 140: 239-45 16 Suto ID, Wilkie LI. The estimation of pyrophosphate in urine with uridine-5' -diphosphoglucose pyrophosphorylase. Clin Chim Acta 1978; 86: 329-32 17 Robertson WG, Scurr DS, Smith A, Orwell RL. The determination of oxalate in urine and urinary calculi by a new ion exchange chromatographic technique. Clin Chim Acta 1982; 126: 91-9 18 Robertson WG, Scurr DS. Prevention of ascorbic acid interference in the measurement of oxalic acid in urine by ion chromatography. Clin Chim Acta 1984; 140: 97-9 19 Werness PG, Brown CM, Smith LH, Finlayson B. EQUIL2: A basic computer program for the calculation of urinary supersaturation. J Uro11985; 134: 1242-4 20 Kohn HN, Hughes RE, McCarthy DI, et al. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: The Pseudo-Gout syndrome II Identification of Crystals. Ann Intern Med 1962; 56: 738-45 21 Roullet IB, Lacour B, Ulmann A, Bally M. Enzymic measurement of urinary pyrophosphate with a centrifugal analyser. Clin Chem 1982; 28: 134-7 22 Akinci M, Esen T, Kocak T, Ozsay C, Tellaloglu S. Role of inhibitor deficiency urolithiasis. I Rationale of urinary magnesium, citrate, pyrophosphate and glycosaminoglycans determinations. Eur Uroll991; 19: 240-3 23 Edwards NA, Russell RGG, Hodgkinson A. The effect of oral phosphate in patients with recurrent renal calculi. Br J Uro11965; 37: 390-8 Accepted for publication 21 January 1992
Downloaded from acb.sagepub.com by guest on June 4, 2016
Ann Clin Biochem 1992; 29: 529-534
Pyrophosphate in synovial fluid and urine and its relationship to urinary risk factors for stone disease N B Roberts, J Dutton, T HelIiweUl, P J N Rothwell- and J P KavanaghFrom the Departments of Chemical Pathology and [Pathology, Royal Liverpool Hospital, Liverpool, £"7 8XW and 2Department of Urology, University Hospital of South Manchester, Manchester M20 8LR, UK
Inorganic pyrophosphate (PP j ) measurement in urine and synovial fluid has been established using the PPj-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 JLmollL, easy to perform and gives results comparable to more complex methods. Daily urinary output of PP j was independently related to both age (P= 0.(014) and sex (P=0·OOO2). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P=O·012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PP j excretion with .urine volume (P = 0·004) and with phosphate excretion (P=0·008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPj. PPj excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PP j concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPj or urate crystals.
SUMMARY.
Additional key phrases: urine phosphate; urine oxalate; hypophosphatasia; arthritis
Inorganic pyrophosphate (PPj) is produced by metabolic pathways involving phosphoribonucleotides. The cumulative daily turnover in humans maybe in the order of kilograms and plasma PP j has a half-life of about 2 min.' Only relatively small amounts of free PP j are actually measurable in plasrna.? urine;' in the mineral of bone" and teeth.' The measurement of an increased excretion of pyrophosphate in urine is the basis for confirmation of the diagnosis of the rare inherited disorder hypophosphatasia." In synovial fluid PPj has been measured in investigations of chondrocalcinosis or pyrophosphate arthropathy (pseudo gout) and other joint disorders.V A low urinary output of PP j could contribute to an increased risk of stone formation as it is well established that PP j can act as an inhibitor of crystallization of various renal stone forming components (of calcium oxalate.P-" brushite and apatite!' and struvite"). Correspondence: Dr N B Roberts.
Russell and Hodgkinson" measured PPj in the urine from stone formers using small ion-exchange columns, but the procedure was laborious and impractical for routine purposes. PPj has also been measured using the enzyme uri dine 5 1_ diphosphoglucose pyrophosphorylase, either with radiolabelled substrates'45: stone stone stone
·
Y .:
..
... :. ....:..........
....:
...
None
Urate
~
PP j
Crystal cantent of synavial fluid FIGURE 3. Pyrophosphate concentrations in synovial fluid from patients with painful, swollen knees; classified by the crystal content of the synovial fluid.
Downloaded from acb.sagepub.com by guest on June 4, 2016
Pyrophosphate in synovial fluid and urine DISCUSSION The analytical procedure described by the manufacturer was modified to enable the simultaneous analysis of up to 16 tests in 1.0 mL volumes. We found the assay was simple to use and had acceptable precision and good recovery for the analysis of PP j in urine or synovial fluid. The arithmetic mean of urinary PP j excretion in healthy controls (33' 5 fLmol/day) compares well with previously reported values, 36' 5 13 and 39' 3 fLmol/day,21 obtained by two different methods. While this is reassuring, it is of limited significance as we observed associations between PP j and age and sex. In addition, the distribution was not symmetrical and the geometric mean (23' 2 fLmol/day) is a better estimate of the central tendency of the spread of results. Schwille et al. ,IS using a specific radioisotopic technique, also note skewed distributions and the importance of age and sex matching. The ranges of PP j in their controls « = 40 years) were 15-98 (men) and 4-46 (women) fLmol/day; our ranges « = 45 years) were very similar at 15-108 (men) and 6-40 (women) fLmol/day. An alternative enzymatic sequence has been used to adapt the method to a centrifugal analyser. 21 However, we have observed that centrifugation of urine can cause a decrease in the absorbance of some urines due to changes in turbidity and not to any chemical reaction. In these cases PP j could be overestimated. The relatively viscous nature of synovial fluid makes it impossible to adapt the assay on this type of sample to a centrifugal analyser, or in fact to any automated system with a fine needle probe. We have therefore adopted the manual procedure for measurement of PP j in urine and synovial fluid. No chemical interferences have been observed from either of these matrices using these enzyme procedures. 7 ,21 There are theoretical and experimental grounds for supposing deficient urinary PP j to be a risk factor for increased urolithogenesisv " but in practice this has not been a common finding. 13,22 This may be due in part to poor age matching of control samples, which if drawn from laboratory personnel would probably have a lower age range and a higher proportion of women than a typical group of stone formers. Both factors would tend to diminish differences between cases and controls, Schwille et al. ,IS who took age and sex into account, found lower PP j urinary output in older stone forming men and younger stone forming women, while we observed this in
533
younger male stone patients. In both studies the general trend is for stone formers to have lower urinary PP j than age and sex matched controls, while the degree of significance attained may be limited by relatively small group numbers. PP j deficiency may be a contributory factor to the aetiology of stone disease, particularly in young women, amongst whom the lowest levels of PP j excretion were found. Calcium oxalate stone formers amongst this group may be most appropriate for supplementation therapy with orthophosphate, which has been shown to increase PPi excretion.P In patients with renal stone disease the 24 h excretion of PP j was correlated only to volume and phosphate excretion (significantly and independently) and not to other important risk factors for stone disease. In practice, the urinary concentration of PP j will be the determining factor in its crystallization inhibitory activity and this decreases with increasing volumes despite the positive association between PP j amount and volume. The usual advice to stone formers to increase their fluid intake may therefore be counter productive if low urinary PP j was a contributory cause of their stone forming diathesis. This may, however, be more than overcome by the decrease in urinary supersaturation. The demonstration of increased urinary PP j excretion has proved most useful in the confirmatory diagnosis of hypophosphatasia. Increased urine PPi outputs were associated with active disease, as reported by Russell. 6 Interestingly, one of the patients had a daughter who did not have active bone disease but had a low plasma alkaline phosphatase (20 fL/L) and a normal PP j excretion (34 fLmol/day). Thus, a low plasma alkaline phosphatase activity is not necessarily associated with active metabolic bone disease in hypophosphatasia and the observation of an elevated urine PPi output may help to establish the onset of bony pathological processes in patients with this disease. Measurement of PP j concentrations in synovial fluid was not helpful in distinguishing pyrophosphate arthropathy from other causes of joint inflammation. Although PP j concentrations were elevated (> 10 fLmol/U) in all four cases associated with PP j crystals, raised levels were also found in 700/0 of samples with urate or no crystal inclusions. The increase in PP j may therefore simply be as a result of increased cellular damage and not related to any particular pathological event.
Downloaded from acb.sagepub.com by guest on June 4, 2016
534
Roberts et al.
The simple and reliable procedure outlined for the measurement of PP j in urine and synovial fluid, without the use of radioisotopes, will ease the routine monitoring and enhance investigations into the role of PP j in the various diseases in which it has been implicated. Acknowledgement We would like to thank Julie A Morris (Department of Statistics, Withington Hospital, Manchester, UK) for advice and for performing the statistical analyses. REFERENCES 1 Dieppe P, Calvert P. Crystals and Joint Disease. London: Chapman and Hall, 1983 2 Fleisch H, Bisaz S. Mechanism of calcification: Inhibitory role of pyrophosphate. Nature 1962; 195: 911
3 Fleisch H, Bisaz S. Isolation from urine of pyrophosphate, a calcification inhibitor. Amer J Physiol 1962; 203: 671-5 4 Perkins HR, Walker P G. The occurrence of pyrophosphate in bone. J Bone Joint Surg 1958; 4OB: 333-6 5 Bisaz S, Russell RGG, Fleisch H. Isolation of inorganic pyrophosphate from bovine and human teeth. Arch Oral Bioi 1968; 13: 683-96 6 Russell RGG. Excretion of pyrophosphate in hypophosphatasia. Lancet 1965; ii: 461-4 7 Altman RD, Muniz OE, Pita IC, Howell DS. Articular chondrocalcinosis microanalysis of pyrophosphate (PPi) in synovial fluid and plasma. Arth Rheum 1973; 16: 171-8 8 Pattrick, M, Hamilton E, Hornby I, Doherty M. Synovial fluid pyrophosphate and nucleoside triphosphate pyrophosphatase: comparison between normal and diseased joints and between inflamed and non-inflamed joints. Ann Rheum Dis 1991; 50: 214-18 9 Robertson WG, Peacock M, Nordin BEC. Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1973; 43: 31-7 10 Grases F, Genesterm G, Conte A, March P, CostaBauza A. Inhibitory effect of pyrophosphate, citrate, magnesium on chondroitin sulphate in calcium oxalate urolithiasis. Br J Urol 1989; 64: 235-7
11 Fleisch H. Inhibitors and promotors of stone formation. Kidney Int 1978; 13: 361-71 12 Mclean RIC, Downey I, Clapham L, Wilson IWL, Nickel IC. Pyrophosphate inhibitors of Proteus mirabilis induced struvite crystalization in vitro. Clin Chim Acta 1991; 20: 107-18 13 Russell ROO, Hodgkinson A. The urinary excretion of inorganic pyrophosphate by normal subjects and patients with renal calculus. Clin Sci 1966;31: 51-62 14 Cheung CP, Suhadolnik RI. Analysis of inorganic pyrophosphate at the picomole level. Anal Biochem 1977; 83: 61-3 15 Schwille PO, Rimenapf G, Wo1fel G, Kohler R. Urinary pyrophosphate in patients with recurrent calcium urolithiasis and in healthy controls: A Reevaluation. J Urol 1988; 140: 239-45 16 Suto ID, Wilkie LI. The estimation of pyrophosphate in urine with uridine-5' -diphosphoglucose pyrophosphorylase. Clin Chim Acta 1978; 86: 329-32 17 Robertson WG, Scurr DS, Smith A, Orwell RL. The determination of oxalate in urine and urinary calculi by a new ion exchange chromatographic technique. Clin Chim Acta 1982; 126: 91-9 18 Robertson WG, Scurr DS. Prevention of ascorbic acid interference in the measurement of oxalic acid in urine by ion chromatography. Clin Chim Acta 1984; 140: 97-9 19 Werness PG, Brown CM, Smith LH, Finlayson B. EQUIL2: A basic computer program for the calculation of urinary supersaturation. J Uro11985; 134: 1242-4 20 Kohn HN, Hughes RE, McCarthy DI, et al. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: The Pseudo-Gout syndrome II Identification of Crystals. Ann Intern Med 1962; 56: 738-45 21 Roullet IB, Lacour B, Ulmann A, Bally M. Enzymic measurement of urinary pyrophosphate with a centrifugal analyser. Clin Chem 1982; 28: 134-7 22 Akinci M, Esen T, Kocak T, Ozsay C, Tellaloglu S. Role of inhibitor deficiency urolithiasis. I Rationale of urinary magnesium, citrate, pyrophosphate and glycosaminoglycans determinations. Eur Uroll991; 19: 240-3 23 Edwards NA, Russell RGG, Hodgkinson A. The effect of oral phosphate in patients with recurrent renal calculi. Br J Uro11965; 37: 390-8 Accepted for publication 21 January 1992
Downloaded from acb.sagepub.com by guest on June 4, 2016
