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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 3951-3957
Copyright © 1998 by The Endocrine Society

Original Studies

Alkaline Phosphatase (EC 3.1.3.1) in Serum Is Inhibited by Physiological Concentrations of Inorganic Phosphate1

Stephen P. Coburn, J. Dennis Mahuren, Manas Jain, Yvonne Zubovic and Jacobo Wortsman

Biochemistry Department, Fort Wayne State Developmental Center (S.P.C., J.D.M., M.J.), Fort Wayne, Indiana 46835; the Department of Mathematical Sciences (Y.Z.), Indiana University and Purdue University, Fort Wayne, Indiana 46805; and the Department of Medicine, Southern Illinois University (J.W.), Springfield, Illinois 62702

Address all correspondence and requests for reprints to: Dr. Stephen P. Coburn, Fort Wayne State Developmental Center, 4900 St. Joe Road, Fort Wayne, Indiana 46835. E-mail: coburn{at}ipfw.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Natural and artificial manipulation of tissue nonspecific alkaline phosphatase activity indicates that pyrophosphate, phosphoethanolamine, and pyridoxal 5'-phosphate are among the natural substrates for this enzyme. Although inorganic phosphate has been recognized as a competitive inhibitor of this enzyme for many years, the influence of phosphate on alkaline phosphatase activity in serum under physiological conditions has not been previously reported. We examined the kinetics of tissue nonspecific alkaline phosphatase from bovine kidney and sera from 49 patients with a wide range of endogenous phosphate concentrations using pyridoxine 5'-phosphate as a substrate at pH 7.4. For the bovine kidney enzyme, the Km was 0.42 ± 0.04 µmol/L, and the Ki for phosphate was 2.4 ± 0.2 µmol/L. Analysis of the kinetics using pyridoxine 5'-phosphate in undiluted serum from 10 subjects with phosphorus ranging from 0.5–2.1 mmol/L and alkaline phosphatase activity ranging from 41–165 nmol/min·mL gave estimates for the Km of 56 ± 11 µmol/L and for the Ki of 540 ± 82 µmol/L for phosphate. This indicates that under physiological conditions alkaline phosphatase activity toward pyridoxine 5'-phosphate is reduced approximately 50% by the normal phosphate concentration and that it will increase or decrease significantly in response to changes in phosphate concentration within the ranges observed clinically.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH assay of alkaline phosphatase activity in serum is a common clinical test, the physiological function of this enzyme remains uncertain. It is a nonspecific phosphomonoesterase usually linked to the external cell surface via glycosyl phosphatidylinositol. Under the proper conditions, catalysis by the enzyme appears to be diffusion limited, making it an almost perfect enzyme (1). In humans there are four genes for this enzyme: intestinal, placental, germ cell, and tissue nonspecific (2). The latter form is posttranslationally modified to differentiate the bone, liver, and kidney isoforms. Decreased activity of the tissue nonspecific enzyme in hypophosphatasia, a genetic disorder, causes significant alterations in the metabolism of phosphoethanolamine, pyrophosphate, and pyridoxal 5'-phosphate, suggesting that these compounds are among the normal substrates for alkaline phosphatase (2). Mathematical equations describing an inverse relationship between alkaline phosphatase activity and pyridoxal 5'-phosphate concentrations have been reported for milk (3) and serum (4). Although maximal enzyme activity is achieved at pH 9–10, the kinetic properties at pH 7.4 are more compatible with physiological concentrations of substrate (5). The enzyme is inhibited uncompetitively by phenylalanine, which is one of the few examples of uncompetitive inhibition of a single substrate enzyme (6). Although inorganic phosphate is well known to be a competitive inhibitor of this enzyme (7), the consequences of this interaction have been examined primarily from the perspective of their potential effect on in vitro assays. For example, Hilliard et al. (8) demonstrated that endogenous phosphate interferes with the determination of alkaline phosphatase in urine and suggested that the wide variation in serum inorganic phosphate concentrations in diseases such as uremia or renal tubular disease might interfere with alkaline phosphatase measurements in serum. The possible effects of endogenous phosphate on phosphatase activity with natural substrates under physiological conditions have not been rigorously evaluated. Fox (9) concluded that because the Ki for inorganic phosphate is 0.6 mmol/L, alkaline phosphatase might be inhibited by phosphate under normal intracellular conditions. The present results indicate that inorganic phosphate is an important physiological regulator of extracellular alkaline phosphatase activity and suggest possible approaches for refining the clinical utility of alkaline phosphatase quantitation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Alkaline phosphatase from bovine kidney (P6680, Sigma Chemical Co., Inc., St. Louis, MO; nominal activity, 72 mmol/h·mg protein in glycine buffer, pH 10.4), bovine placenta (Sigma P3765; nominal activity, 13 µmol/h·mg solid preparation in glycine buffer, pH 10.4), and bovine intestinal mucosa (Sigma P6772; nominal activity, 162 mmol/h·mg protein in diethanolamine buffer, pH 9.8) were obtained from Sigma Chemical Co. as was pyridoxal 5'-phosphate. Pyridoxine 5'-phosphate was synthesized according to the method described by Peterson and Sober (10).

The activity of the purified enzymes toward pyridoxine 5'-phosphate and pyridoxal 5'-phosphate was measured using modifications of the procedures of Ubbink and Schnell (11). The assay mixture contained 800 µL buffer (50 mmol/L triethanolamine, pH 7.4) containing 5 mmol/L magnesium chloride, 100 µL enzyme, 10 µL sodium phosphate at various concentrations adjusted to pH 7.4, and 100 µL pyridoxal 5'-phosphate or pyridoxine 5'-phosphate at various concentrations. Assays at pH 10.0 were conducted similarly, except that the buffer was 0.1 mol/L Tris. After incubation for 6 min at 37 C, the reaction was stopped by the addition of 1 mL 5% trichloroacetic acid, extracted for 1 min with 4 mL peroxide-free diethyl ether on a vortex mixer, and centrifuged at 1000 x g for 10 min. The aqueous layer was analyzed for pyridoxine (or pyridoxal) using cation exchange high performance liquid chromatography (12). Because of the minute amount of protein precipitate, it was not necessary to centrifuge before adding the ether.

Analysis of phosphatase activity in serum used 200 µL undiluted serum (or a 1:100 dilution in 50 mmol/L triethanolamine buffer, pH 7.4), 10 µL sodium phosphate at various concentrations adjusted to pH 7.4, and 10 µL substrate in the range of 5–50 µmol/L. After incubation for 10 min at 37 C, the reaction was stopped with 1 mL 5% trichloroacetic acid and centrifuged at 1000 x g for 10 min. The supernatant was transferred to a second tube, extracted, and analyzed as described above.

Clinical samples of sera selected for a range of phosphorus concentrations (29 men, 52 ± 19 yr; 20 women, 59 ± 19 yr) were obtained from a pathology laboratory. Phosphorus was determined by a modification of the Fiske and Subbarow method (Paramax, Dade International, Newark, DE). Clinical assay of alkaline phosphatase was performed at pH 10.3, using p-nitrophenyl phosphate in a modification of the method of Bowers and McComb (Paramax).

Kinetic data were analyzed using SAAM II (SAAM Institute, Seattle, WA). This program can determine the single best fit to multiple sets of reaction conditions. The data for purified enzymes were fit to curves rather than to linear transformations (13). The data for whole serum were analyzed using Dixon plots of 1/v vs. [I] (14). This permits estimation of kinetic parameters even though all samples contain inhibitor. Three inhibitor concentrations at each of three substrate concentrations were examined in 10 subjects. The statistical characteristics of the kinetic constants for this population of 10 datasets were evaluated using an extended multiple studies analysis (15).

An equation for predicting pyridoxine phosphate phosphatase activity in undiluted serum (with a substrate concentration of 5 µmol/L) from inorganic phosphate concentrations and the Bowers and McComb alkaline phosphatase assay (16) was obtained by multiple regression of the Michaelis-Menten equation for competitive inhibition using the mean Km and Ki values obtained above in undiluted serum and allowing the maximum velocity (Vmax) to adjust to give the best fit to the 45 data points. Four points were omitted because the activity was so high that the reaction was not linear with 5 µmol/L substrate. A nomogram was developed from the resulting equation (17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Substrate inhibition was observed with all enzymes, but was lowest in the intestinal form (Table 1Go and Figs. 1–3GoGoGo). The bell-shaped curves produced by plotting velocity vs. the logarithm of the substrate concentration are consistent with substrate inhibiting the reaction by binding at a second site (18). The Km for pyridoxine 5'-phosphate at pH 7.4 for the bovine enzymes ranged from 0.19–1.6 µmol/L (Table 1Go). The Km for pyridoxine 5'-phosphate was similar to the Km for pyridoxal 5'-phosphate, although Vmax was lower with pyridoxal 5'-phosphate (Table 1Go and Fig. 4Go). As substrate inhibition was reduced with pyridoxine 5'-phosphate and nonspecific protein binding was minimized, pyridoxine 5'-phosphate was used as the routine substrate. When analyzed for the simultaneous best fit to all four curves in Fig. 5Go, assuming pure competitive inhibition, the parameter estimates for the bovine kidney enzyme were 0.42 ± 0.04 µmol/L for Km, 2.4 ± 0.2 µmol/L for Ki, and 74 ± 1 µmol/min·mg for Vmax. These data showed a slight, but consistent, reduction in Vmax with increasing phosphate concentration. Adding a term to compensate for uncompetitive inhibition reduced the residual sum of squares of the curvilinear solution approximately 40%, but had little effect on the values for Km (0.37 ± 0.03 µmol/L), Ki (2.2 ± 0.2 µmol/L), or Vmax (74 ± 1 µmol/min·mg) because these parameters are defined primarily by earlier segments of the curve. The Ki for the uncompetitive term, which was designed to include both substrate and phosphate concentrations was 117 ± 60 µmol/L. A Lineweaver-Burk plot confirms that competitive inhibition is the primary effect (Fig. 6Go).


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  		Table 1. Kinetic properties of bovine alkaline phosphatases including substrate inhibition

 


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  		Figure 1. Kinetics of bovine placental alkaline phosphatase at pH 7.4 using pyridoxine 5'-phosphate as the substrate.

 


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  		Figure 2. Kinetics of bovine intestinal alkaline phosphatase at pH 7.4 using pyridoxine 5'-phosphate as the substrate.

 


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  		Figure 3. Kinetics of bovine kidney alkaline phosphatase at pH 7.4 using pyridoxine 5'-phosphate as the substrate. (The data at a substrate concentration of 10 µmol/L were excluded when fitting the curve.)

 


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  		Figure 4. Kinetics of bovine kidney alkaline phosphatase at pH 7.4 using pyridoxal 5'-phosphate as the substrate.

 


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  		Figure 5. Phosphate inhibition of bovine kidney alkaline phosphatase at pH 7.4 using pyridoxine 5'-phosphate as the substrate. Phosphate concentration ([Pi]) = 0 ({blacktriangleup}), 3 µmol/L (•), 6 µmol/L ({blacktriangledown}), and 12 µmol/L ({blacksquare}).

 


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  		Figure 6. Lineweaver-Burk plot of the data in Fig. 5Go. Phosphate concentration ([Pi]) = 0 ({blacktriangledown}), 3 µmol/L (•), 6 µmol/L ({blacksquare}), and 12 µmol/L ({blacktriangleup}).

 
Although the results from purified enzymes under artificial conditions are useful for examining some of the properties of the enzyme, physiological pH and ionic environment may pose some functional constraints. Standard clinical assays for serum alkaline phosphatase are based on highly diluted serum, which would minimize the inhibitory effects of endogenous inorganic phosphate. To approximate the in vivo situation most reliably, we measured pyridoxine phosphate phosphatase activity in undiluted serum in 49 specimens with varying endogenous inorganic phosphate concentrations. We found that when calculated on the basis of activity per mL serum, the pyridoxine phosphate phosphatase activity in the diluted samples was approximately 7-fold greater than that in undiluted serum (Table 2Go). This is clear evidence that phosphatase activity in serum is severely inhibited under normal physiological conditions of pH and substrate concentration. Further evidence of the effect of endogenous inorganic phosphate in undiluted serum was the significant negative correlation between endogenous phosphate and phosphatase activity in undiluted serum at pH 7.4 and the lack of significant correlation between endogenous phosphate and phosphatase activity in diluted serum at pH 7.4 or pH 10 (Table 3Go). The correlation between pyridoxine phosphatase activity at pH 7.4 in samples diluted 1:100 and routine clinical assay at pH 10 was 0.95. This demonstrates that when the influences of inhibitors are minimized by dilution, assays at either pH 10 or pH 7.4 give a good estimate of the relative total activity. The high correlation of the activity at pH 7.4 with the activity at pH 10 also indicates that the pyridoxine phosphatase activity at pH 7.4 is due primarily to alkaline phosphatase with little contribution from acid phosphatase. In contrast to that high degree of correlation between the two methods using diluted serum with different substrates at different pH, the correlation between the results of either method with diluted serum and undiluted serum was less than 0.5 (Table 3Go). Although the correlations are still statistically significant, such an r value has little predictive power. Therefore, activity measured under normal assay conditions does not necessarily reflect true in vivo activity. Further evidence that it is the activity in undiluted serum that may be most physiologically relevant is the fact that the correlation between serum pyridoxal 5'-phosphate and phosphatase activity at pH 7.4 in undiluted serum was statistically significant, whereas the correlation with activity in diluted serum was not (Table 3Go).


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  		Table 2. Phosphatase activity in serum (n = 49)

 

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  		Table 3. Correlations between various data

 
Based on the above results we concluded that the most valid method of estimating pyridoxine phosphate phosphatase activity in vivo is to run the assay in undiluted serum. A Dixon plot of 1/v against [I] permits determination of kinetic constants even though no sample is completely free of inhibitor (14) (Fig. 7Go). Results in undiluted serum from 10 subjects evaluated using the multiple studies procedure of Lyne et al. (15) gave population means of Km for pyridoxine phosphate of 56 ± 11 µmol/L, Vmax of 8.5 ± 1.5 nmol/mL·min, and Ki for inorganic phosphate of 540 ± 82 µmol/L. Presumably, the increased values compared to the values in Table 1Go for purified enzymes reflect various interactions with serum components.



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  		Figure 7. Dixon plot using pyridoxine 5'-phosphate (PNP) in undiluted serum PNP = 5 µmol/L (•), 10 µmol/L ({blacktriangledown}), and 50 µmol/L ({blacksquare}).

 
As current clinical assays are not designed for testing undiluted serum, we investigated various multiple regression approaches to predicting the pyridoxine phosphate phosphatase activity from the common clinical assays for inorganic phosphate and alkaline phosphatase. The most appropriate relationship was derived from the usual equation for competitive inhibition yielding the following equation:

(1)
where Y is the predicted pyridoxine phosphate phosphatase activity in undiluted serum (nanomoles per L/min) with a substrate concentration of 5 µmol/L as used in our assay, Pi is inorganic phosphorus (milligrams per dL), and ALP is alkaline phosphatase activity determined by the method of Bowers and Mccomb (units per L or micromoles per L/min). The mean ratio of observed/predicted values was 1.1 ± 0.3, with a range of 0.5–1.6. In addition to the three-dimensional presentation in Fig. 8Go, the relationship can be converted into a nomogram (Fig. 9Go).



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  		Figure 8. Relationship among clinical alkaline phosphatase data, inorganic phosphate, and pyridoxine phosphate phosphatase activity in undiluted serum. Points are measured values. The curve indicates pyridoxine phosphate phosphatase values predicted by inserting clinical alkaline phosphatase and inorganic phosphate measurements into Eq I.

 


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  		Figure 9. Nomogram for predicting pyridoxine phosphate phosphatase in undiluted serum from usual clinical measurements of inorganic phosphate and alkaline phosphatase.

 
As the samples were obtained from patients with a wide variety of clinical conditions, it might be suggested that the relationships between alkaline phosphatase and inorganic phosphate could be influenced by other factors not considered here. Therefore, we examined the ratio of the activity in undiluted serum before and after adding phosphate to increase the concentration 2 mmol/L using the equation:

(2)
Using the ratio facilitates comparisons between patients with different Vmax values. The addition of phosphate to serum generally followed the predicted relationship for a competitive inhibitor at phosphorus concentrations spanning the range of 0.4–3.4 mmol/L (1–10 mg/dL) and clinical alkaline phosphatase activity ranging from 39–353 U/L (Fig. 10Go). Therefore, the underlying clinical substrate did not appear to significantly alter the phosphate-phosphatase relationship. This analysis also provided an opportunity to obtain additional verification of the Km and Ki values estimated earlier from 10 Dixon plots. The use of ratios prevented the estimation of absolute values without some constraints. Therefore, we used the mean and SDs obtained from the Dixon plots as Bayesian parameters for Km and Ki in the SAAMII analysis. The best fit to 46 data points from undiluted serum using Eq II estimated the Km for pyridoxine phosphate as 52 ± 11 µmol/L and the Ki for inorganic phosphate as 735 ± 57 µmol/L compared with 56 and 540 based on 10 direct measurements. Considering the complexity of these systems, we consider this to be acceptable concordance.



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  		Figure 10. Ratio of pyridoxine phosphate phosphatase activity in undiluted serum before and after increasing the phosphate concentration by 2 mmol/L as described by Eq II.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The optimum pH for alkaline phosphatase assay varies with the substrate concentration. Using glycerophosphate as the substrate, Ross et al. (5) reported an optimum pH of 9.13 at 10 mg/mL, but 7.35 at 3 µg/mL. The values reported here are similar to those reported previously (Table 4Go). The Km of 0.19 µmol/L for the bovine kidney enzyme with pyridoxal 5'-phosphate is compatible with the observed bovine plasma pyridoxal phosphate concentration of 0.3 µmol/L (19).


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  		Table 4. Reported estimates of Km and Ki for inorganic phosphate for pyridoxal (pyridoxine) phosphate phosphatase activity

 
Several lines of evidence suggest that alkaline phosphatase is the enzyme primarily responsible for pyridoxine phosphate phosphatase activity in serum. The lack of alkaline phosphatase activity in hypophosphatasia is associated with markedly increased pyridoxal 5'-phosphate concentrations in plasma (20). Inactivating the tissue nonspecific alkaline phosphatase gene in mice also leads to similar abnormalities of vitamin B-6 metabolism (21). Lumeng and Li (22) concluded that pyridoxal phosphate hydrolase activity in rat liver was identical with alkaline phosphatase. The subcellular fraction with the most activity was the nuclear fraction, which contained the plasma membrane fragments. This is consistent with the evidence that tissue nonspecific alkaline phosphatase is an ectoenzyme. In the current study, pyridoxine phosphate phosphatase activity at pH 7.4 in diluted serum was highly correlated (r = 0.95; P < 0.001) with the clinical assay for alkaline phosphatase using p-nitrophenyl phosphate at pH 10.3. Therefore, we conclude that the pyridoxine phosphate phosphatase activity in serum is due to alkaline phosphatase.

Other workers have commented on substrate inhibition of pyridoxine phosphatase (23, 24). We found it to have a detectable effect at concentrations as low as 10 µmol/L. The possibility that phosphate exerts a mixed inhibition effect has also been mentioned previously. Fernley and Walker (25) noted that although phosphate inhibition was predominantly competitive, there was a mixed component in calf intestinal alkaline phosphatase. However, for the conditions used in our study, omitting the uncompetitive inhibition term had little effect on the results. Fernley and Walker also noted that although 1 mmol/L magnesium ion stimulated the hydrolysis of 4-methylumbelliferyl phosphate, it caused over 50% inhibition of the hydrolysis of pyrophosphate and ATP. As the magnesium ion concentration in serum is approximately 1 mmol/L, phosphomonoesterase activity is probably predominant. They reported that the Ki for inorganic phosphate was 16.5 µmol/L compared with a Km of 21.5 µmol/L at pH 9.25. The Km and Ki values increased by 100-fold as the pH was increased from 8.0 to 10. At pH 8.0, both Km and Ki were approximately 1.4 µmol/L, which is quite comparable to our Km value of 1.6 µmol/L for bovine intestinal enzyme at pH 7.4 with pyridoxine 5'-phosphate as the substrate. Bowers and McComb (26) concluded that dilution of the serum in their assay would reduce the phosphate concentration in the final reaction mixture to 20 µmol/L, which caused less than 1% inhibition under their assay conditions.

The observations reported here raise an interesting question about the clinical significance of alkaline phosphatase measurements. As a result of the phosphate inhibition effect, pyridoxine phosphate phosphatase activity in undiluted serum was similar in patients with widely differing alkaline phosphatase activity in the standard clinical assay. For example, pyridoxine phosphate phosphatase activity in the undiluted serum of a patient with a phosphorus concentration of 2.1 mg/dL and alkaline phosphatase activity of 41 U/L was 249 nmol/L·min compared with 276 in another patient with a phosphorus concentration of 10.5 mg/dL and alkaline phosphatase activity of 252 U/L. Thus, even though the clinical assay detected a 6-fold difference in alkaline phosphatase activity, the functional activity under physiological conditions was similar, probably as a result of phosphate inhibition. This raises the possibility that some renal patients with high serum phosphate and normal alkaline phosphatase activity might have functional hypophosphatasia, which could contribute to renal osteodystrophy. The effect of phosphate might also explain the lack of correlation between alkaline phosphatase activity and pyridoxal 5'-phosphate concentrations reported by others (27). The observation of very low pyridoxal 5'-phosphate concentrations in the plasma of patients with hypophosphatemic rickets (28) may be an example of the effect of phosphate on vitamin B-6 metabolism. The researchers (28) suggested that the low pyridoxal 5'-phosphate concentrations were the result of increased alkaline phosphatase activity. However, most of the alkaline phosphatase values reported (28) were within the normal range for children (16). Mean pyridoxal 5'-phosphate concentrations in normal 13- to 14-yr-old adolescents have been reported to be approximately 40 nmol/L (29), even though this developmental stage is associated with high phosphatase activity (16). Inorganic phosphate is also high. Therefore, it seems likely that the main factor in the decreased pyridoxal phosphate concentrations in hypophosphatemic rickets may be low phosphate rather than high alkaline phosphatase.

As renal failure is frequently associated with increased serum phosphate and some increase in alkaline phosphatase activity, the net effect on pyridoxal phosphate hydrolysis will depend on the relative magnitude of the changes in these two parameters. Spannuth et al. (30) reported that plasma clearance of pyridoxal 5'-phosphate increased in uremic patients, suggesting that alkaline phosphatase was the dominant factor. However, the elevated clearances reported by Spannuth et al. were similar to the control values reported by Lui et al. (31), indicating that further data are needed to clarify all of the factors affecting the determination of plasma clearance.

The standard clinical assay for alkaline phosphatase using alkaline conditions with high concentrations of artificial substrates and highly diluted serum presumably measures Vmax under those particular conditions. Such measurements provide useful diagnostic data. Perhaps the information from this assay is an indicator of the amount of alkaline phosphatase being released from cells. As alkaline phosphatase is an ectoenzyme, the activity in serum may be relatively insignificant compared to the bound activity. However, recognition that the functional activity of free (and presumably bound) alkaline phosphatase toward its natural substrates in serum is reduced over 50% by normal extracellular phosphate concentrations and will increase or decrease significantly in response to variations in inorganic phosphate within the clinically observed range may lead to refinements in methodology and/or new paradigms for interpreting alkaline phosphatase data. Even if there is no clinical advantage, this information increases our understanding of the physiological role of alkaline phosphatase, particularly its regulation of vitamin B-6 metabolism.


   Acknowledgments
 
The authors appreciate the comments and suggestions of M. P. Whyte.


   Footnotes
 
1 This work was supported in part by Grants 91–37200-6181 and 95–37200-1703 from the USDA National Research Initiative Competitive Grant Program. A preliminary report was presented at Experimental Biology ’97 (FASEB J. 11:A178, 1997). Back

Received May 12, 1998.

Revised August 5, 1998.

Accepted August 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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