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Activities of bone and liver alkaline phosphatases in serum in health and disease
209
Cfinica Chimica Acta, 80 (1977) 209-220 @ Elsevier/North-Holland Biomedical Press
CCA 8900
ACTIVITIES OF BONE AND LIVER ALKALINE IN SERUM IN HEALTH AND DISEASE
KATRINE
B. WHITAKER,
PHOSPHATASES
L.G. WHITBY * and D.W. MOSS
Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS (U.K.) (Received
April 29th,
1977)
summary A heat-inactivation method for determining absolute activities of liver and bone alkaline phosphatases in serum has been applied extensively in routine diagnosis. Values for each isoenzyme in healthy individuals of different ages are reported together with results obtained in various diseases. Data from normal subjects show that bone alkaline phosphatase contributes about half the total alkaline phosphatase activity in adults. Liver phosphatase shows a slight increase with age. The method is also able to detect reliably the presence of carcinoplacental isoenzymes.
Introduction The value of characterizing alkaline phosphatase isoenzymes in serum as a diagnostic aid is now well established in clinical enzymology [ 11. However, except for the determination of intestinal and placental isoenzymes, the methods used are generally of a qualitative or semi-quantitative nature and results are frequently expressed in terms of a relative change in the activity of one or other component. of serum alkaline phosphatase. Qualitative assessments of the relative contributions of liver and bone alkaline phosphatases to the total activity in serum are often made and are of considerable diagnostic value. The usefulness of bone and liver isoenzymes of alkaline phosphatase in diagnosis should be even further enhanced by quantitative measurement. Moss and Whitby [2] described a procedure by which the selective inactivation of alkaline phosphatase isoenzymes by heat [3] can be used to obtain quantitative data on the activity of liver phosphatase present in serum samples. This procedure has
* Permanent address: EH3 9YW (U.K.).
University Department
of Clinical Chemistry. Royal Infirmary, Edinburgh
210
now been used in this laboratory for two years to determine the alkaline phosphatase isoenzyme composition of samples of serum from patients. In addition, the activities of liver and bone alkaline phosphatase isoenzymes in the sera of apparently healthy individuals of various ages have been determined. The availability of these reference ranges makes it possible to interpret correctly the absolute activities of liver and bone alkaline phosphatases which can be obtained by this method. Materials and methods Thermal inactivation of serum alkaline phosphatase at 56°C was carried out by the procedure and with the precautions described by Moss and Whitby [ 21, with the following modifications: 100 ~1 of each serum was introduced into a pre-heated tube, 50 1.11was withdrawn to a tube pre-cooled in ice at 15 min and the tube with the remaining 50 ~1 was removed to ice at 25 min. Total and residual alkaline phosphatase activity was determined with 20 ~1 of unheated and the two heated samples respectively. The method used for determination of initial and residual alkaline phospha~ activities at 37°C was that recommended by the Scandinavian Committee on Enzymes [4] with ~-nitrophenyl phosphate as the substrate and diethanolamine as the buffer. Measurements were made in the LKB Produkter AB 8600 reaction-rate analyzer. The apparent half-life, t, (s), of liver alkaline phosphatase and its activity (I.U./l) in serum were calculated as follows: t,,, = 180.6/(log
log activity
a - log b)
= 2.5 log a - 1.5 log b
(i) (ii)
where Q and b are the residual activities after 15 and 25 min incubation, respectively. Alternatively, the activity of liver alkaline phosphatase can be derived from a plot of the residual activities against time on semi-logarithmic graph paper by extrapolating the line joining the two points to zero time [2]. The half-life of liver phosphatase can also be derived from this graph. The activity of bone alkaline phosphatase is obtained by subtracting the liver phosphatase activity from the initial alkaline phosphatase activity of the sample. Samples of pooled serum introduced into each batch of alkaline phosphatase activity determinations gave an estimate of long-term between-batch reproducibility of -+2.7% C.V. at the 400 I.U./l level for the assay of activity. Duplicate estimations of the isoenzyme composition of samples analyzed on separate occasions indicated that reproducibility of the heat-inactivation method for liver phosphatase was +5.8% C.V. (mean 453 I.U./l; n = 15) and for bone phosphatase *10.2% C.V. (mean 281 I.U./l; n = 15). Replicate determinations on one serum gave +8.0% C.V. for liver phosphatase at a level of 204 I.U./l (n = 5) and &9.2% C.V. for bone phosphatase at a level of 205 I-U./l (n = 5). Serum-to-serum variations in half-life occur [ 5 1, and this fact forms the main indication for the use of the present method, in which these variations are taken into account. However, in any batch of estimations, the average half-life will tend towards the overall mean value found for a large number of sera. Therefore, the constancy of average half-life from batch to batch is an indica-
211
tion of consistency of analytical performance. Two types of specimens, discussed below, can distort the average half-life if the batch contains a significant proportion of such specimens. If such specimens are excluded, large variations of the average half-life from the mean value may indicate analytical malfunction, such as gross variation in incubation temperature or timing. The mean of 82 batch-means from batches of average size 8, excluding sera subject to systematic errors, was 451 s 2 18 S.D. Therefore, if samples are analyzed in batches, analytical error should be suspected if the batch-mean half-life deviates by more than 2 S.D. from this value. Small ~tween-Bach v~ations in temperature affect the half-life but not the measured activity of liver phosphatase since each residual activity is altered in the same proportion. Alkaline phosphatase isoenzyme zones were separated by vertical polyacrylamide gel electrophoresis [ 11. Serum samples from non-fasting healthy subjects were obtained from hospital and laboratory workers and blood donors. Samples from patients were submitted to the laboratory for routine diagnostic investigation. Results The mean half-life of liver alkaline phosphatase in 110 normal sera and 461 sera from patients with various diseases was 452 s + 55 S.D.. This value excludes results from samples in which the activity of bone alkaline phosphatase greatly exceeded that of liver phosphatase on electrophoresis, or if a pronounced intestinal alkaline phosphatase zone was present. This value is nearly identical with that of 456 s + 90 S.D. found by Whitby and Moss [5] by analysis of multipoint heat-inactivation cubes. The apparent half-life of liver phosphatase was ~dependent of the level of activity of this isoenzyme from 6 to 5230 I.U./l (Fig. 1). Values for half-life of liver phospha~ which diverged markedly from
s j
700
J 600 5f 0
.
“a5cm .-8 54 Y m t 300 .L _i F 200 c i: I
50
I
100
I
I
200
500
I
loo0
I.
2ccKl
I
5ooc
Liver alkaline phosphatase activity (I.U./I) Fig. 1. Raiationshin between measursd blf-life of liver alkaline phosphatase and the activity of liver phosphatese in sera from 559 patients and 110 normal individuals. A, sera which contain a very high proportion of bone alkaline phosphatase, in which this method underestimates half-life. (One serum with a liver phoaphatase h&f-life of SO6 sand 6 I.U./l Ever phospbatase activity has been omitted).
212
the mean were found in only a minority of sera and these fell generally into two categories. The first category consisted of sera which were seen to contain very high proportions of bone alkaline phosphatase on electrophoretic analysis, and which were characterized by an apparent half-life of liver phosphatase more than 1 S.D. below the average value. The low apparent half-life in such samples is due to the elevation of the residual activity at 15 min by bone phosphatase which has survived heating at 56°C; thus, the calculated liver phosphatase activity is correspondingly overestimated and may appear falsely to exceed the normal range. Although the residual activity at 15 min contains a significant amount of bone phosphatase activity in these cases, the contribution of bone phosphatase to the residual activity at 25 min is very small indeed; e.g. the average half-life of bone phosphatase [5] shows that only 1 I.U./l of an initial 10 000 I.U./l of the bone isoenzyme would be present after 25 min at 56°C. Thus, a more accurate value of liver alkaline phosphatase activity can be obtained by a calculation using the residual activity at 25 min and the mean halflife of liver phosphatase: log (liver activity)
= log (activity
at 25 min) +
1500 log 2 t,
(iii)
Values for liver phosphatase activity thus obtained are not as reliable as those for sera in which corrections are unnecessary, because with this correction no allowance is made for variation in half-life from serum to serum. The modified calculation was applied to 38 sera shown by electrophoresis to contain very high proportions of the bone isoenzyme and giving apparent half-lives for liver phosphatase which were less than 400 s (i.e. more than 1 SD. below the mean). In 20 of these sera (Table I), the application of this correction converted an apparently elevated value for liver phosphatase into a more accurate normal or only slightly raised value. In the second category of samples with apparently anomalous half-lives of liver alkaline phosphatase, values more than 2 SD. above the average were observed (Table II). The apparent half-lives in these sera were due to the presence of placental or placental-like (Regan) alkaline phosphatases in varying amounts. The activity of these heat-stable alkaline phosphatase isoenzymes can be measured by determining the residual activity after heating at 65°C for 30 min. If this activity is subtracted from the total activity and the residual activities at 15 and 25 min, the corrected residual activities can be substituted in Eqns. i and ii and calculation of the half-life of liver phosphatase and the activities of the liver and bone isoenzymes made in the usual way. The validity of this procedure is shown by the values of half-life thus obtained (Table II). Variation of total serum alkaline phosphatase activity with age in 110 apparently healthy individuals is similar to that found in other studies (Fig. 2). There is a decline in the third decade from the higher levels found in younger subjects, and a slight upward trend is apparent in the older age groups. The average activity in males is slightly higher than in females, but the difference is not statistically significant for subjects over 25 years of age. However, for subjects of 17 to 25 years this difference is statistically significant (P< 0.01). Corresponding values for the individual isoenzymes confirm that bone alkaline phosphatase is
213 TABLE I Activities of liver and bone alkaline phosphatases in sera containing very high proportions of bone alkaline phosphatase, before and after applicationof a modified calculation (see text). The effect of correction in these cases ia to reduce apparently elevated activities of liver alkaline phoilphatase to normal or nearnormal levels, and to raise correspondingly the values for bone phosphatase activity. Apparent trh of phosphatase (8)
Apparent liver phosphatase activity U.W-0
308 313 316 322 326 327 338 341 341 360 361 366 369 375 37s 37s 394 397 397 398
654 351 295 287 149 17s 170 376 375 188 212 195 236 260 126 206 199 2413 181 215
liver
03 80 26 14 9 13 15 54 03 73 74 18 69 69 14 26 32 65 75 47
* Activity still
sIi&tIy
I
25
Corrected
liver pllospllatase activity fI.U./l) 185 127 108 111 60 73 77 168 173 103 117 111 138 153 78 127 139 168 129 154
*
* *
* * * * *
Apparent bone phosphate=
Corrected bone phosphatase
activitv
activity
c1.u.nt
tl.u./l)
934 1494 674 911 806 626 537 1432 840 503 377 497 884 300 325 731 577 684 266 499
1303 1718 761 1087 895 732 630 1640 1042 688 472 581 982 897 373 810 637 661 318 560
raised after correction.
I
35
I 45
I 55
I 65
Age (years) Fig. 2. Variation of total alkaline phosphatase activity with age in 110 normal individuals: 0, males; 0, females. The Central line rePreSent mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
109 140 246 245 430 1604 419
cI.u.n,
activity
Total aIkaIine phosphatasc
Apparent t4/nof liver phosphatase (s)
661 657 4120 4405 4261 814 9930
Placental phosphatasc activity (I.U./l)
2 7 77 I33 154 189 294 519 431 538 534 471 424 476
True t4kof liver phosphatase (5)
Correct Iiver phosphatase aCtiVity (I.U./I) 61 93 46 55 109 1415 83
Apparent liver phosphatase activity (I.U. P) 58 74 104 115 212 1130 338
correct bone phosphatase activity &U./l) 46 40 123 107 167 0 42
Apparent bone phosphatase activity (I.U./l) 51 66 142 130 218 474 81
Activities of IIver and bone aIkaIine phosphatases in some sera with apparent haif-lives of liver phosphatase more than 2 S.D. above the mean value. Uncorrected actkvities and activities calculated after correction for the effect of placental-type aIkaIine phosphatase (measured as residual activity after incubation at 65’0 are shown. This correction also akws the true half-life of Liver phosphatase to be determined. -
TABLE II
215
15
25
35
45
55
65
Age (years) Fig. 3. Variation of bone alkaline phosphatase activity with age in 110 normal individuals: l, males; 0, females. The central line represents mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
responsible for the elevated total activity in young people and the activity of bone alkaline phosphatase remains considerable in middle and later life, making up approximately half the total activity (Fig. 3). Again, males have slightly more bone phosphatase than females and this difference is statistically significant (P < 0.02) at ages below 25, but not in older people. Liver alkaline phosphatase shows a slight upward trend with age and this appears to account for the slight increase in total activity in older age groups (Fig. 4). There is no significant difference in values for liver phosphatase between men and women. Neither negative nor positive correlation exists between the amounts of bone and liver phosphatase in normal sera. Thus, the activities of the two isoenzymes in serum appear to be under independent control. Of the 110 normal subjects, 33 had detectable amounts of intestinal alkaline phosphatase on electrophoresis. Sixteen of these had considerable amounts of
Age (years) Fig. 4. Variation of liver alhaline phosphatase activity with age in 110 normal individuals: l, msles; 0, females. The central line represents mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
216 TABLE
III
Reference ranges for different and bone alkaline phosphatases TOtal activity (1.U.D)
Age (years)
<17 17-19 20-2s 30-39 40-49 50-59 6-9
age groups for total alkaline phosphatase in serum derived from Figs. 2-4.
*
*
60-270 60-230 60-210 60-210 70-220 70-230
* Values for these age-groups
Liver phosphatase activity (I.U./I) 20-100 20-110 20-110 30-l 20 30-130 3(t-140 30-l 50 were obtained
activity
and for activities
of liver
Bone phosphatase activity u.u./u 20-190 lo-160 5-120 5-120 10-110 20-110 by extrapolation.
this isoenzyme compared with the accompanying liver phosphatase while the remaining 17 had only traces of intestinal phosphatase. The apparent liver phosphatase activity for all sera containing intestinal phosphatase was on average slightly higher than for sera without the detectable intestinal isoenzyme. However, this difference was statistically significant (P < 0.02) only for sera containing a considerable proportion of intestinal phosphatase. Reference ranges for activities of bone and liver alkaline phosphatases appropriate to various age groups derived by inspection of the data of Figs. 2-4 are indicated in Table III. These ranges correspond approximately to 97% confidence limits. When the activities of liver or bone alkaline phosphatase are plotted against total alkaline phosphatase activity in serum, several categories of patients can be distinguished (Figs. 5, 6). In the majority of patients with elevated total alkaline phosphatase, this is due to a corresponding increase in one or other isoenzyme, i.e., either liver or bone disease is present. More significant and useful results from a diagnostic point of view are represented by those cases in which the activities of both isoenzymes are increased concurrently, or in which one or other isoenzyme is elevated although the total activity is within normal limits. Elevation of both isoenzymes occurred in patients with malignant disease, malabsorption, and renal failure due to various causes. In a few patients, both isoenzymes were raised as a result of coexisting, but causally unrelated, disorders of liver and bone, such as Paget’s disease accompanying miscellaneous liver diseases. The activities of liver or bone alkaline phosphatase were raised in a small but significant proportion of patients with total alkaline phosphatase activities within normal limits. About half of the elevated activities of liver phosphatase in this category occurred in children. Amongst the adult patients with raised liver phosphatase activity but normal total activities, diagnoses were made of minimal liver disease due to various causes, e.g., alcoholism, infection, or malignant infiltration. A few cases were encountered in which a slightly raised activity of bone alkaline phosphatase was accompanied by normal total activity and
217
1
I
I
I
a5
1
5
I
10
J
50
Total alkaline phosphatase activity (multiples of upper limit of norfrd)
of liver alkaline phosphatase as a function of total alkaline phoaphatase activity in sera fkom 535 patients. Each activity is expressed as a multiple ofthe upper limM of the normal range appropdate to the patient’s age. F&s 5. Activity
*
.I. . fi
. I.. . .;::. .. ..-.t
.
.
Total olkalu-8ephosphatase activity (multiples of upper limit of normal) Fig. 6. Activity of bone alkaline phosphetase BP P function of total alkaline phosphatese activity in sere from 635 potients. Each activity is expressed as 8 multipIe of the upper limit of the normal range approp-
riate to the petient’s age.
218
in these cases minimal osteomalacia or Paget’s disease of bone were thought account for the increase in bone phosphatase.
to
Discussion This study confirms the conclusions of Whitby and Moss [ 51 that the heatstability characteristics of liver alkaline phosphatase are not quantitatively identical in all sera, although the magnitude of these variations is not such as to abolish the difference in stability between liver and bone alkaline phosphatases in a given serum. The measurement of residual activities after two chosen periods of heat inactivation [2] takes into account the variation in heat stability from sample to sample in calculating the isoenzyme composition of each sample. In this respect the present method is superior to those in which calculation is based on a single me~urement of residual activity except when a gross excess of bone alkaline phosphatase falsely lowers the apparent half-life of liver alkaline phosphatase. Such sera can be recognized by their electrophoretic pattern, allowing an appropriate correction to be made. Electrophoresis of serum samples also allows samples with a pronounced zone of intestinal alkaline phosphatase to be recognized, and this technique should always be used in conjunctjon with heat inactivation. The heat-stability characteristics of the intestinal isoenzyme are indistin~ishable from those of liver phosphatase in the heat-inactivation procedure, and any intestinal alkaline phosphatase present in the sample is therefore included in the calculated activity of liver phosphatase [ 21. Significant interference by intestinal alkaline phosphatase will be less frequent if samples are obtained from fasting patients. Different procedures, such as inhibition with L-phenyl~anine [ 61, are available for the quantitation of intestinal alkaline phosphatase if required. Placental or placental-like alkaline phosphatases are less easily recognized on electrophoresis because of their variable mobilities, but their effect on the apparent half-life of liver phosphatase in the present method is a sensitive indication of the presence of these isoenzymes. The method described here is simpler than the manual [5] or automated [7] determination of full heat-inactivation curves for each serum sample, but shares their advantage over single-period inactivation procedures of providing results that are independent of small variations in inactivating temperature from one batch of analyses to another. Our observation that the total alkaline phosphat~e activity in the serum of healthy adults is composed of approximately equal activities of liver and bone phosphatases agrees with other reports [7,8]. In a group of young adults (1830 years of age), Statland et al. [9] found that 58% of the total alkaline phosphatase activity was due to the bone isoenzyme. Our average value for the contribution of bone isoenzyme in this age group is 57%. The upward trend in liver phosphatase activity in serum with age noted in this work supports the similar conclusion reached by Sharland [lo] on the basis of visual assessment of electrophoretic patterns. Quantitative measurements of liver and bone alkaline phosphatases show their greatest diagnostic value in the investigation of liver function in children and in the detection of coexisting liver and bone lesions, particularly in malignant disease.
219
References 1 2 3 4 5 6 7 8 9 10
Moss, D.W. (1975) Enzyme 20.20-34 Moss. D.W. and Whitby. L.G. (1975) Clin. Chim. Acta 61. 63-71 Moss, D.W. and King, E.J. (1962) Biochem. J. 84.192-195 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry ogy (1974) Stand. J. Clin. Lab. Invest. 33. 291-306 Whitby. L.G. and Moss, D.W. (1975) CIin. Cbim. Acta 59. 361-367 Fishman. W.H.. Green, S. and Inglis. N.I. (1963) Nature 198.685-686 PetitClerc. C. (1976) Clin. Chem. 22, 4248 .%&x% J.. Volek, V. and Kol&, J. (1976) Clin. Chim. Acta 69. l-9 Statland, B.E.. Nishi. H.H. and Young. D.S. (1972) Clin. Chem. 18, 1468-1474 Sharland. D.E. (1974) Clin. Chim. Acta 56. 187-198
and Clinical Physiol-
Cfinica Chimica Acta, 80 (1977) 209-220 @ Elsevier/North-Holland Biomedical Press
CCA 8900
ACTIVITIES OF BONE AND LIVER ALKALINE IN SERUM IN HEALTH AND DISEASE
KATRINE
B. WHITAKER,
PHOSPHATASES
L.G. WHITBY * and D.W. MOSS
Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS (U.K.) (Received
April 29th,
1977)
summary A heat-inactivation method for determining absolute activities of liver and bone alkaline phosphatases in serum has been applied extensively in routine diagnosis. Values for each isoenzyme in healthy individuals of different ages are reported together with results obtained in various diseases. Data from normal subjects show that bone alkaline phosphatase contributes about half the total alkaline phosphatase activity in adults. Liver phosphatase shows a slight increase with age. The method is also able to detect reliably the presence of carcinoplacental isoenzymes.
Introduction The value of characterizing alkaline phosphatase isoenzymes in serum as a diagnostic aid is now well established in clinical enzymology [ 11. However, except for the determination of intestinal and placental isoenzymes, the methods used are generally of a qualitative or semi-quantitative nature and results are frequently expressed in terms of a relative change in the activity of one or other component. of serum alkaline phosphatase. Qualitative assessments of the relative contributions of liver and bone alkaline phosphatases to the total activity in serum are often made and are of considerable diagnostic value. The usefulness of bone and liver isoenzymes of alkaline phosphatase in diagnosis should be even further enhanced by quantitative measurement. Moss and Whitby [2] described a procedure by which the selective inactivation of alkaline phosphatase isoenzymes by heat [3] can be used to obtain quantitative data on the activity of liver phosphatase present in serum samples. This procedure has
* Permanent address: EH3 9YW (U.K.).
University Department
of Clinical Chemistry. Royal Infirmary, Edinburgh
210
now been used in this laboratory for two years to determine the alkaline phosphatase isoenzyme composition of samples of serum from patients. In addition, the activities of liver and bone alkaline phosphatase isoenzymes in the sera of apparently healthy individuals of various ages have been determined. The availability of these reference ranges makes it possible to interpret correctly the absolute activities of liver and bone alkaline phosphatases which can be obtained by this method. Materials and methods Thermal inactivation of serum alkaline phosphatase at 56°C was carried out by the procedure and with the precautions described by Moss and Whitby [ 21, with the following modifications: 100 ~1 of each serum was introduced into a pre-heated tube, 50 1.11was withdrawn to a tube pre-cooled in ice at 15 min and the tube with the remaining 50 ~1 was removed to ice at 25 min. Total and residual alkaline phosphatase activity was determined with 20 ~1 of unheated and the two heated samples respectively. The method used for determination of initial and residual alkaline phospha~ activities at 37°C was that recommended by the Scandinavian Committee on Enzymes [4] with ~-nitrophenyl phosphate as the substrate and diethanolamine as the buffer. Measurements were made in the LKB Produkter AB 8600 reaction-rate analyzer. The apparent half-life, t, (s), of liver alkaline phosphatase and its activity (I.U./l) in serum were calculated as follows: t,,, = 180.6/(log
log activity
a - log b)
= 2.5 log a - 1.5 log b
(i) (ii)
where Q and b are the residual activities after 15 and 25 min incubation, respectively. Alternatively, the activity of liver alkaline phosphatase can be derived from a plot of the residual activities against time on semi-logarithmic graph paper by extrapolating the line joining the two points to zero time [2]. The half-life of liver phosphatase can also be derived from this graph. The activity of bone alkaline phosphatase is obtained by subtracting the liver phosphatase activity from the initial alkaline phosphatase activity of the sample. Samples of pooled serum introduced into each batch of alkaline phosphatase activity determinations gave an estimate of long-term between-batch reproducibility of -+2.7% C.V. at the 400 I.U./l level for the assay of activity. Duplicate estimations of the isoenzyme composition of samples analyzed on separate occasions indicated that reproducibility of the heat-inactivation method for liver phosphatase was +5.8% C.V. (mean 453 I.U./l; n = 15) and for bone phosphatase *10.2% C.V. (mean 281 I.U./l; n = 15). Replicate determinations on one serum gave +8.0% C.V. for liver phosphatase at a level of 204 I.U./l (n = 5) and &9.2% C.V. for bone phosphatase at a level of 205 I-U./l (n = 5). Serum-to-serum variations in half-life occur [ 5 1, and this fact forms the main indication for the use of the present method, in which these variations are taken into account. However, in any batch of estimations, the average half-life will tend towards the overall mean value found for a large number of sera. Therefore, the constancy of average half-life from batch to batch is an indica-
211
tion of consistency of analytical performance. Two types of specimens, discussed below, can distort the average half-life if the batch contains a significant proportion of such specimens. If such specimens are excluded, large variations of the average half-life from the mean value may indicate analytical malfunction, such as gross variation in incubation temperature or timing. The mean of 82 batch-means from batches of average size 8, excluding sera subject to systematic errors, was 451 s 2 18 S.D. Therefore, if samples are analyzed in batches, analytical error should be suspected if the batch-mean half-life deviates by more than 2 S.D. from this value. Small ~tween-Bach v~ations in temperature affect the half-life but not the measured activity of liver phosphatase since each residual activity is altered in the same proportion. Alkaline phosphatase isoenzyme zones were separated by vertical polyacrylamide gel electrophoresis [ 11. Serum samples from non-fasting healthy subjects were obtained from hospital and laboratory workers and blood donors. Samples from patients were submitted to the laboratory for routine diagnostic investigation. Results The mean half-life of liver alkaline phosphatase in 110 normal sera and 461 sera from patients with various diseases was 452 s + 55 S.D.. This value excludes results from samples in which the activity of bone alkaline phosphatase greatly exceeded that of liver phosphatase on electrophoresis, or if a pronounced intestinal alkaline phosphatase zone was present. This value is nearly identical with that of 456 s + 90 S.D. found by Whitby and Moss [5] by analysis of multipoint heat-inactivation cubes. The apparent half-life of liver phosphatase was ~dependent of the level of activity of this isoenzyme from 6 to 5230 I.U./l (Fig. 1). Values for half-life of liver phospha~ which diverged markedly from
s j
700
J 600 5f 0
.
“a5cm .-8 54 Y m t 300 .L _i F 200 c i: I
50
I
100
I
I
200
500
I
loo0
I.
2ccKl
I
5ooc
Liver alkaline phosphatase activity (I.U./I) Fig. 1. Raiationshin between measursd blf-life of liver alkaline phosphatase and the activity of liver phosphatese in sera from 559 patients and 110 normal individuals. A, sera which contain a very high proportion of bone alkaline phosphatase, in which this method underestimates half-life. (One serum with a liver phoaphatase h&f-life of SO6 sand 6 I.U./l Ever phospbatase activity has been omitted).
212
the mean were found in only a minority of sera and these fell generally into two categories. The first category consisted of sera which were seen to contain very high proportions of bone alkaline phosphatase on electrophoretic analysis, and which were characterized by an apparent half-life of liver phosphatase more than 1 S.D. below the average value. The low apparent half-life in such samples is due to the elevation of the residual activity at 15 min by bone phosphatase which has survived heating at 56°C; thus, the calculated liver phosphatase activity is correspondingly overestimated and may appear falsely to exceed the normal range. Although the residual activity at 15 min contains a significant amount of bone phosphatase activity in these cases, the contribution of bone phosphatase to the residual activity at 25 min is very small indeed; e.g. the average half-life of bone phosphatase [5] shows that only 1 I.U./l of an initial 10 000 I.U./l of the bone isoenzyme would be present after 25 min at 56°C. Thus, a more accurate value of liver alkaline phosphatase activity can be obtained by a calculation using the residual activity at 25 min and the mean halflife of liver phosphatase: log (liver activity)
= log (activity
at 25 min) +
1500 log 2 t,
(iii)
Values for liver phosphatase activity thus obtained are not as reliable as those for sera in which corrections are unnecessary, because with this correction no allowance is made for variation in half-life from serum to serum. The modified calculation was applied to 38 sera shown by electrophoresis to contain very high proportions of the bone isoenzyme and giving apparent half-lives for liver phosphatase which were less than 400 s (i.e. more than 1 SD. below the mean). In 20 of these sera (Table I), the application of this correction converted an apparently elevated value for liver phosphatase into a more accurate normal or only slightly raised value. In the second category of samples with apparently anomalous half-lives of liver alkaline phosphatase, values more than 2 SD. above the average were observed (Table II). The apparent half-lives in these sera were due to the presence of placental or placental-like (Regan) alkaline phosphatases in varying amounts. The activity of these heat-stable alkaline phosphatase isoenzymes can be measured by determining the residual activity after heating at 65°C for 30 min. If this activity is subtracted from the total activity and the residual activities at 15 and 25 min, the corrected residual activities can be substituted in Eqns. i and ii and calculation of the half-life of liver phosphatase and the activities of the liver and bone isoenzymes made in the usual way. The validity of this procedure is shown by the values of half-life thus obtained (Table II). Variation of total serum alkaline phosphatase activity with age in 110 apparently healthy individuals is similar to that found in other studies (Fig. 2). There is a decline in the third decade from the higher levels found in younger subjects, and a slight upward trend is apparent in the older age groups. The average activity in males is slightly higher than in females, but the difference is not statistically significant for subjects over 25 years of age. However, for subjects of 17 to 25 years this difference is statistically significant (P< 0.01). Corresponding values for the individual isoenzymes confirm that bone alkaline phosphatase is
213 TABLE I Activities of liver and bone alkaline phosphatases in sera containing very high proportions of bone alkaline phosphatase, before and after applicationof a modified calculation (see text). The effect of correction in these cases ia to reduce apparently elevated activities of liver alkaline phoilphatase to normal or nearnormal levels, and to raise correspondingly the values for bone phosphatase activity. Apparent trh of phosphatase (8)
Apparent liver phosphatase activity U.W-0
308 313 316 322 326 327 338 341 341 360 361 366 369 375 37s 37s 394 397 397 398
654 351 295 287 149 17s 170 376 375 188 212 195 236 260 126 206 199 2413 181 215
liver
03 80 26 14 9 13 15 54 03 73 74 18 69 69 14 26 32 65 75 47
* Activity still
sIi&tIy
I
25
Corrected
liver pllospllatase activity fI.U./l) 185 127 108 111 60 73 77 168 173 103 117 111 138 153 78 127 139 168 129 154
*
* *
* * * * *
Apparent bone phosphate=
Corrected bone phosphatase
activitv
activity
c1.u.nt
tl.u./l)
934 1494 674 911 806 626 537 1432 840 503 377 497 884 300 325 731 577 684 266 499
1303 1718 761 1087 895 732 630 1640 1042 688 472 581 982 897 373 810 637 661 318 560
raised after correction.
I
35
I 45
I 55
I 65
Age (years) Fig. 2. Variation of total alkaline phosphatase activity with age in 110 normal individuals: 0, males; 0, females. The Central line rePreSent mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
109 140 246 245 430 1604 419
cI.u.n,
activity
Total aIkaIine phosphatasc
Apparent t4/nof liver phosphatase (s)
661 657 4120 4405 4261 814 9930
Placental phosphatasc activity (I.U./l)
2 7 77 I33 154 189 294 519 431 538 534 471 424 476
True t4kof liver phosphatase (5)
Correct Iiver phosphatase aCtiVity (I.U./I) 61 93 46 55 109 1415 83
Apparent liver phosphatase activity (I.U. P) 58 74 104 115 212 1130 338
correct bone phosphatase activity &U./l) 46 40 123 107 167 0 42
Apparent bone phosphatase activity (I.U./l) 51 66 142 130 218 474 81
Activities of IIver and bone aIkaIine phosphatases in some sera with apparent haif-lives of liver phosphatase more than 2 S.D. above the mean value. Uncorrected actkvities and activities calculated after correction for the effect of placental-type aIkaIine phosphatase (measured as residual activity after incubation at 65’0 are shown. This correction also akws the true half-life of Liver phosphatase to be determined. -
TABLE II
215
15
25
35
45
55
65
Age (years) Fig. 3. Variation of bone alkaline phosphatase activity with age in 110 normal individuals: l, males; 0, females. The central line represents mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
responsible for the elevated total activity in young people and the activity of bone alkaline phosphatase remains considerable in middle and later life, making up approximately half the total activity (Fig. 3). Again, males have slightly more bone phosphatase than females and this difference is statistically significant (P < 0.02) at ages below 25, but not in older people. Liver alkaline phosphatase shows a slight upward trend with age and this appears to account for the slight increase in total activity in older age groups (Fig. 4). There is no significant difference in values for liver phosphatase between men and women. Neither negative nor positive correlation exists between the amounts of bone and liver phosphatase in normal sera. Thus, the activities of the two isoenzymes in serum appear to be under independent control. Of the 110 normal subjects, 33 had detectable amounts of intestinal alkaline phosphatase on electrophoresis. Sixteen of these had considerable amounts of
Age (years) Fig. 4. Variation of liver alhaline phosphatase activity with age in 110 normal individuals: l, msles; 0, females. The central line represents mean activities and the upper and lower lines have been drawn to include approximately 97% of the points.
216 TABLE
III
Reference ranges for different and bone alkaline phosphatases TOtal activity (1.U.D)
Age (years)
<17 17-19 20-2s 30-39 40-49 50-59 6-9
age groups for total alkaline phosphatase in serum derived from Figs. 2-4.
*
*
60-270 60-230 60-210 60-210 70-220 70-230
* Values for these age-groups
Liver phosphatase activity (I.U./I) 20-100 20-110 20-110 30-l 20 30-130 3(t-140 30-l 50 were obtained
activity
and for activities
of liver
Bone phosphatase activity u.u./u 20-190 lo-160 5-120 5-120 10-110 20-110 by extrapolation.
this isoenzyme compared with the accompanying liver phosphatase while the remaining 17 had only traces of intestinal phosphatase. The apparent liver phosphatase activity for all sera containing intestinal phosphatase was on average slightly higher than for sera without the detectable intestinal isoenzyme. However, this difference was statistically significant (P < 0.02) only for sera containing a considerable proportion of intestinal phosphatase. Reference ranges for activities of bone and liver alkaline phosphatases appropriate to various age groups derived by inspection of the data of Figs. 2-4 are indicated in Table III. These ranges correspond approximately to 97% confidence limits. When the activities of liver or bone alkaline phosphatase are plotted against total alkaline phosphatase activity in serum, several categories of patients can be distinguished (Figs. 5, 6). In the majority of patients with elevated total alkaline phosphatase, this is due to a corresponding increase in one or other isoenzyme, i.e., either liver or bone disease is present. More significant and useful results from a diagnostic point of view are represented by those cases in which the activities of both isoenzymes are increased concurrently, or in which one or other isoenzyme is elevated although the total activity is within normal limits. Elevation of both isoenzymes occurred in patients with malignant disease, malabsorption, and renal failure due to various causes. In a few patients, both isoenzymes were raised as a result of coexisting, but causally unrelated, disorders of liver and bone, such as Paget’s disease accompanying miscellaneous liver diseases. The activities of liver or bone alkaline phosphatase were raised in a small but significant proportion of patients with total alkaline phosphatase activities within normal limits. About half of the elevated activities of liver phosphatase in this category occurred in children. Amongst the adult patients with raised liver phosphatase activity but normal total activities, diagnoses were made of minimal liver disease due to various causes, e.g., alcoholism, infection, or malignant infiltration. A few cases were encountered in which a slightly raised activity of bone alkaline phosphatase was accompanied by normal total activity and
217
1
I
I
I
a5
1
5
I
10
J
50
Total alkaline phosphatase activity (multiples of upper limit of norfrd)
of liver alkaline phosphatase as a function of total alkaline phoaphatase activity in sera fkom 535 patients. Each activity is expressed as a multiple ofthe upper limM of the normal range appropdate to the patient’s age. F&s 5. Activity
*
.I. . fi
. I.. . .;::. .. ..-.t
.
.
Total olkalu-8ephosphatase activity (multiples of upper limit of normal) Fig. 6. Activity of bone alkaline phosphetase BP P function of total alkaline phosphatese activity in sere from 635 potients. Each activity is expressed as 8 multipIe of the upper limit of the normal range approp-
riate to the petient’s age.
218
in these cases minimal osteomalacia or Paget’s disease of bone were thought account for the increase in bone phosphatase.
to
Discussion This study confirms the conclusions of Whitby and Moss [ 51 that the heatstability characteristics of liver alkaline phosphatase are not quantitatively identical in all sera, although the magnitude of these variations is not such as to abolish the difference in stability between liver and bone alkaline phosphatases in a given serum. The measurement of residual activities after two chosen periods of heat inactivation [2] takes into account the variation in heat stability from sample to sample in calculating the isoenzyme composition of each sample. In this respect the present method is superior to those in which calculation is based on a single me~urement of residual activity except when a gross excess of bone alkaline phosphatase falsely lowers the apparent half-life of liver alkaline phosphatase. Such sera can be recognized by their electrophoretic pattern, allowing an appropriate correction to be made. Electrophoresis of serum samples also allows samples with a pronounced zone of intestinal alkaline phosphatase to be recognized, and this technique should always be used in conjunctjon with heat inactivation. The heat-stability characteristics of the intestinal isoenzyme are indistin~ishable from those of liver phosphatase in the heat-inactivation procedure, and any intestinal alkaline phosphatase present in the sample is therefore included in the calculated activity of liver phosphatase [ 21. Significant interference by intestinal alkaline phosphatase will be less frequent if samples are obtained from fasting patients. Different procedures, such as inhibition with L-phenyl~anine [ 61, are available for the quantitation of intestinal alkaline phosphatase if required. Placental or placental-like alkaline phosphatases are less easily recognized on electrophoresis because of their variable mobilities, but their effect on the apparent half-life of liver phosphatase in the present method is a sensitive indication of the presence of these isoenzymes. The method described here is simpler than the manual [5] or automated [7] determination of full heat-inactivation curves for each serum sample, but shares their advantage over single-period inactivation procedures of providing results that are independent of small variations in inactivating temperature from one batch of analyses to another. Our observation that the total alkaline phosphat~e activity in the serum of healthy adults is composed of approximately equal activities of liver and bone phosphatases agrees with other reports [7,8]. In a group of young adults (1830 years of age), Statland et al. [9] found that 58% of the total alkaline phosphatase activity was due to the bone isoenzyme. Our average value for the contribution of bone isoenzyme in this age group is 57%. The upward trend in liver phosphatase activity in serum with age noted in this work supports the similar conclusion reached by Sharland [lo] on the basis of visual assessment of electrophoretic patterns. Quantitative measurements of liver and bone alkaline phosphatases show their greatest diagnostic value in the investigation of liver function in children and in the detection of coexisting liver and bone lesions, particularly in malignant disease.
219
References 1 2 3 4 5 6 7 8 9 10
Moss, D.W. (1975) Enzyme 20.20-34 Moss. D.W. and Whitby. L.G. (1975) Clin. Chim. Acta 61. 63-71 Moss, D.W. and King, E.J. (1962) Biochem. J. 84.192-195 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry ogy (1974) Stand. J. Clin. Lab. Invest. 33. 291-306 Whitby. L.G. and Moss, D.W. (1975) CIin. Cbim. Acta 59. 361-367 Fishman. W.H.. Green, S. and Inglis. N.I. (1963) Nature 198.685-686 PetitClerc. C. (1976) Clin. Chem. 22, 4248 .%&x% J.. Volek, V. and Kol&, J. (1976) Clin. Chim. Acta 69. l-9 Statland, B.E.. Nishi. H.H. and Young. D.S. (1972) Clin. Chem. 18, 1468-1474 Sharland. D.E. (1974) Clin. Chim. Acta 56. 187-198
and Clinical Physiol-