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Serum trehalase: assay and normal values
CLINICA CHIMICA ACTA
SERUM
E. VAN
TREHALASE:
349
ASSAY
AND
NORMAL
VALUES
HANDEL
Entomological Research Center, Vero Beach, Florida (U.S.A.) (Received
March zqth,
1970)
SUMMARY
A method is presented for the evaluation of serum trehalase. It depends on the determination of glucose, produced from trehalose after incubation with serum. Glucose is determined without deproteinization, either with glucose oxidase, or with o-toluidine. In normal blood donors, glucose production ranged from o--1000 ,ug/h (o-o.09 pmole/min) per ml of serum. The trehalase level was fairly constant in the individual, and no age or sex difference was observed.
INTRODUCTION
Trehalase is the only disaccharidase present in normal human serum1*2,3. As serum enzymes are usually derived from the tissue where the enzyme functions, abnormal values may be connected with disease of that tissue. Trehalase is present to a small extent in liver, and in large concentrations in the cortex of the kidneyl++. It has been localized in the convoluted tubules by histochemical technique9. Previous work has shown distinct differences of serum trehalase among individualsly”93. Prior to its evaluation in the clinic, the fluctuation in a healthy population has been determined, using a method which seems equally suitable for the clinical laboratory. METHOD
Bz$er solution (0.1 M; pH = 6.0). Dissolve 12 g NaH,PO,.H,O and 3.3 g Na,HPO, .7H,O in I 1 water. Substrate solution (IS mM). Dissolve 5.7 g trehalose dihydrate in I 1 buffer solution. Glucose Yeagent. Dissolve 25 mg glucose oxidase (trehalase-free), zg mg horseradish peroxidase, and 50 mg dianisidine.zHCl in I 1 buffer solution. When kept in the refrigerator, this solution can be used for several weeks. Alternative glucose reagent. Dissolve 1.5 g thiourea in g4o ml glacial acetic acid, and add 60 ml o-toluidine. The solution is stable for many months. This reagent is developed by Hultman7. C&z. Chim. Acta, 2g (1970) 349-353
350
VAN
HANDEL
Stan&d stock solution. Dissolve 500 mg glucose in 30-40 ml water, then dilute to IOO ml with ethanol. This standard keeps indefinitely. Glucose standard. On the day of use, dilute I ml stock solution to IO ml with water;
50 ~1 = 25 pg. Reagent sources. Glucose oxidase (pure, trehalase-free) and horseradish peroxidase were purchased from Worthington Biochemical Corporation, Freehold, New Jersey; trehalose from Nutritional Biochemicals Corporation, Cleveland, Ohio; dianisidine and o-toluidine from Eastman Organic Chemicals, Rochester, New York. All other chemicals were of reagent grade quality.
PROCEDURE
Enzymatic glucose fwoductio~z. Place 0.1 ml serum in each of two 10x75 mm test tubes. Add I ml buffer solution to one tube (control) and I ml substrate solution to the other tube (trehalase). Add about 25 ,ul or one drop of toluene and stopper with a cork. Incubate overnight at 37’. Enzymatic glucose measurement. Place 50 ,ul aliquots of the buffer solution (reagent blank), the glucose standard, the control tube, and the trehalase tube in IO x75 mm spectrophotometer tubes, add 2 ml glucose oxidase reagent and read the absorbance after about 15 min at room temperature at 450 m,u. If the absorbance exceeds 0.80, it is advisable to repeat the reaction with a smaller than 50 ,~61aliquot. The aliquots can best be taken with a microsyringe. (Alternative) chemical glucose measurement. Place the 50 ,ul aliquots in 16 x IOO mm test tubes, add 2 ml of the o-toluidine reagent, heat at 100' for IO min, and read in IO x 75 mm spectrophotometer tubes at 630 m,u. Calculation The trehalase activity, expressed as ,ug of glucose produced hour per ml serum at 37’ equals: (absorbance
trehalase tube-absorbance
hrs incubated
at 37O x absorbance
control tube)
x
from trehalose
per
25 x 220
glucose standard
The factor 25 represents the glucose standard (25 ,ug).The factor 220 represents the 50 ~1 aliquot, taken from the 1.1 ml enzyme dilution (= 0.0045 ml serum). To convert from pg glucose/ml/h to mpmole glucose/ml/min, the values should be multiplied with 0.093. RESULTS
The frequency distribution of serum trehalase, assayed in 250 healthy blood donors is represented in Table I. The range is from 25-900 units with a median value of 200 to 300. About 7% of the population had values below 50, close to the lowest limit of detectability. There is no clear age trend, because approximately the same range and average values were found in donors from 18 to 60 years old (Table II). The average value of 285 units for 200 males was only slightly higher than the average of 260 for 50 females. C&n. Chim.
Acta,
zg (1970) x49-35.1
3.51
SERUM TREHALASE TABLE
I
DISTRIBUTION TYehalase
OFTREHALASEIN
units*
O-49 P-99 IOO-rgg 200-299
OFHEALTHYBLOOD
of donovs
48 67 30 24 29 9 3 3
700-799 Boo-~000 * ,ug glucose produced per
hour per
DONORS
Frequency(%) 7.5 7 19 27 12
I9 18
300-399 400-499 500-599 60~699
TABLE
THESERUM
Number
10
11.5 3.5 I I mlof
serum at 37'.
II
TREHALASE
AS AFUNCTION
Nuwzbe4
OFAGE
of
TrehaEase
_-._ a-47
50
O-700
260
28-37 38-47 48-60
68 89 4'
0-650 o-go0 O-700
300 290 270
I
The variability of the trehalase activity among donors suggests that the enzyme might vary considerably within the same individual. To investigate this, serum trehalase was determined in 21 donors, 3 to 6 months after the first assay. The correlation between the first and the second value (r = 0.86) was highly significant (Table III). This indicates that trehalase activity is fairly representative of the individual. The enzyme is quite stable and showed little loss of activity when serum was frozen for many months at -15~. Up to 48 h after incubation at 37”, glucose was produced proportional to time. It is possible to speed up the determination, at least for high trehalase values, by incubating a more concentrated serum for a shorter time period, e.g. 0.5 ml serum+o.5 ml substrate for 4 to 6 h. The pH optima is 5.6-6.0. The substrate concentration (IS mM) is above saturation concentration. When the logarithm of the enzyme activity was plotted against I/T (Arrhenius plot), a linear relation was obtained from IO to 37”. From the slope of this plot, the Qlo was calculated to be 1.8, and the activation energy gooo Cal/mole. DISCUSSION
In 40 serum samples, glucose was estimated both with glucose-oxidase and with o-toluidine. The differences were within 5%. This small variability is quite acceptable in view of the wide range of serum trehalase values in the sample of the normal population. The assay takes place in a very dilute serum (0.0045 ml), whereas the biogenic control tube corrects for any absorbance not related to the enzyme. It therefore Clin. Chim. Acta,
q
(1970)
349-353
VAN HANDEL
352 TABLE
III
REINVESTIGAIION OF THE SAME DONOR,
First
200
50 200
value
80
200
220
300 230
350
270
300 300 500
210
80 100
120
250 250 150 4.50 600 100 IO0
150
200
400 200
600 550 15”
180
LATER
170
400
200
MONTHS
Same donor 3-6 mods later
420 440
50 3.50 50 25
SEVERAL
160
appeared unnecessary to prepare a protein-free filtrate, even in the presence of turbidity, hemolysis or high bilirubin values. Several specimens with less than 50 trehalase units were mixed with equal parts of a standard serum of 900 units. The mixed sera always had the calculated average trehalase value, indicating that the low values were not due to inhibition of trehalase. Glucose standards added to the control tubes were always quantitatively recovered, both with the glucose oxidase and with the o-toluidine reagents. When the glucose oxidase reagent is free of trehalase, it is not necessary to use a substrate blank, because trehalose does not hydrolyze non-enzymatically. Trehalase of human serum has been recently investigated by Courtois and his students1p2. Their normal values are within the range of those in Table I. Much higher values than those reported here for the human are found in the serum of the rabbit, the squirrel, and the armadillo3. Trehalose, the only substrate known to react with trehalase, is the main blood sugar of many insects and some other invertebrates. So far, it has not been demonstrated in any vertebrate animal. The hypothesis that trehalase plays a role in the transport of glucose through the kidney is under current investigation536. ACKNOWLEDGEMENTS
Serum of blood donors was kindly provided by Dr. L. E. McHenry, director of pathology of the Brevard Hospital. The trehalase determinations were done by Mrs. Joye Chauncey. This work was supported by a grant (AI 09363) from the National Institutes of Health.
Clin. Chim. Acta, 29 (1970) 349-353
SERUM TREHALASE
353
REFERENCES I J. E. COURTOISAND J. F. DEMELIER,Bull. Sec. Chim. Biol., 48 (1966) 277. 2 J.C. GEORGET, Recherches SW la Trehalase du serum humain. Thesis, University of Paris series U-No. 446 (1966-1967).
VAN HANDEL, Cow@. Biochem. Physiol., 26 (1968) 561. 4 I.S. LUKOMSKAYA AND G. I.TARASOVA,Biokhimia, 30(1965) =JE. VAN HANDEL, .%ience, 163 (1969)1075. 6 W. GROSSMAN AND B. SACKTOR,Science, 161 (1968)571. 7 E. HULTMAN, Nature, 183 (1959)108. 3 E.
95.
Cl&z.Chim. Acta, zg (1970) 349-353
SERUM
E. VAN
TREHALASE:
349
ASSAY
AND
NORMAL
VALUES
HANDEL
Entomological Research Center, Vero Beach, Florida (U.S.A.) (Received
March zqth,
1970)
SUMMARY
A method is presented for the evaluation of serum trehalase. It depends on the determination of glucose, produced from trehalose after incubation with serum. Glucose is determined without deproteinization, either with glucose oxidase, or with o-toluidine. In normal blood donors, glucose production ranged from o--1000 ,ug/h (o-o.09 pmole/min) per ml of serum. The trehalase level was fairly constant in the individual, and no age or sex difference was observed.
INTRODUCTION
Trehalase is the only disaccharidase present in normal human serum1*2,3. As serum enzymes are usually derived from the tissue where the enzyme functions, abnormal values may be connected with disease of that tissue. Trehalase is present to a small extent in liver, and in large concentrations in the cortex of the kidneyl++. It has been localized in the convoluted tubules by histochemical technique9. Previous work has shown distinct differences of serum trehalase among individualsly”93. Prior to its evaluation in the clinic, the fluctuation in a healthy population has been determined, using a method which seems equally suitable for the clinical laboratory. METHOD
Bz$er solution (0.1 M; pH = 6.0). Dissolve 12 g NaH,PO,.H,O and 3.3 g Na,HPO, .7H,O in I 1 water. Substrate solution (IS mM). Dissolve 5.7 g trehalose dihydrate in I 1 buffer solution. Glucose Yeagent. Dissolve 25 mg glucose oxidase (trehalase-free), zg mg horseradish peroxidase, and 50 mg dianisidine.zHCl in I 1 buffer solution. When kept in the refrigerator, this solution can be used for several weeks. Alternative glucose reagent. Dissolve 1.5 g thiourea in g4o ml glacial acetic acid, and add 60 ml o-toluidine. The solution is stable for many months. This reagent is developed by Hultman7. C&z. Chim. Acta, 2g (1970) 349-353
350
VAN
HANDEL
Stan&d stock solution. Dissolve 500 mg glucose in 30-40 ml water, then dilute to IOO ml with ethanol. This standard keeps indefinitely. Glucose standard. On the day of use, dilute I ml stock solution to IO ml with water;
50 ~1 = 25 pg. Reagent sources. Glucose oxidase (pure, trehalase-free) and horseradish peroxidase were purchased from Worthington Biochemical Corporation, Freehold, New Jersey; trehalose from Nutritional Biochemicals Corporation, Cleveland, Ohio; dianisidine and o-toluidine from Eastman Organic Chemicals, Rochester, New York. All other chemicals were of reagent grade quality.
PROCEDURE
Enzymatic glucose fwoductio~z. Place 0.1 ml serum in each of two 10x75 mm test tubes. Add I ml buffer solution to one tube (control) and I ml substrate solution to the other tube (trehalase). Add about 25 ,ul or one drop of toluene and stopper with a cork. Incubate overnight at 37’. Enzymatic glucose measurement. Place 50 ,ul aliquots of the buffer solution (reagent blank), the glucose standard, the control tube, and the trehalase tube in IO x75 mm spectrophotometer tubes, add 2 ml glucose oxidase reagent and read the absorbance after about 15 min at room temperature at 450 m,u. If the absorbance exceeds 0.80, it is advisable to repeat the reaction with a smaller than 50 ,~61aliquot. The aliquots can best be taken with a microsyringe. (Alternative) chemical glucose measurement. Place the 50 ,ul aliquots in 16 x IOO mm test tubes, add 2 ml of the o-toluidine reagent, heat at 100' for IO min, and read in IO x 75 mm spectrophotometer tubes at 630 m,u. Calculation The trehalase activity, expressed as ,ug of glucose produced hour per ml serum at 37’ equals: (absorbance
trehalase tube-absorbance
hrs incubated
at 37O x absorbance
control tube)
x
from trehalose
per
25 x 220
glucose standard
The factor 25 represents the glucose standard (25 ,ug).The factor 220 represents the 50 ~1 aliquot, taken from the 1.1 ml enzyme dilution (= 0.0045 ml serum). To convert from pg glucose/ml/h to mpmole glucose/ml/min, the values should be multiplied with 0.093. RESULTS
The frequency distribution of serum trehalase, assayed in 250 healthy blood donors is represented in Table I. The range is from 25-900 units with a median value of 200 to 300. About 7% of the population had values below 50, close to the lowest limit of detectability. There is no clear age trend, because approximately the same range and average values were found in donors from 18 to 60 years old (Table II). The average value of 285 units for 200 males was only slightly higher than the average of 260 for 50 females. C&n. Chim.
Acta,
zg (1970) x49-35.1
3.51
SERUM TREHALASE TABLE
I
DISTRIBUTION TYehalase
OFTREHALASEIN
units*
O-49 P-99 IOO-rgg 200-299
OFHEALTHYBLOOD
of donovs
48 67 30 24 29 9 3 3
700-799 Boo-~000 * ,ug glucose produced per
hour per
DONORS
Frequency(%) 7.5 7 19 27 12
I9 18
300-399 400-499 500-599 60~699
TABLE
THESERUM
Number
10
11.5 3.5 I I mlof
serum at 37'.
II
TREHALASE
AS AFUNCTION
Nuwzbe4
OFAGE
of
TrehaEase
_-._ a-47
50
O-700
260
28-37 38-47 48-60
68 89 4'
0-650 o-go0 O-700
300 290 270
I
The variability of the trehalase activity among donors suggests that the enzyme might vary considerably within the same individual. To investigate this, serum trehalase was determined in 21 donors, 3 to 6 months after the first assay. The correlation between the first and the second value (r = 0.86) was highly significant (Table III). This indicates that trehalase activity is fairly representative of the individual. The enzyme is quite stable and showed little loss of activity when serum was frozen for many months at -15~. Up to 48 h after incubation at 37”, glucose was produced proportional to time. It is possible to speed up the determination, at least for high trehalase values, by incubating a more concentrated serum for a shorter time period, e.g. 0.5 ml serum+o.5 ml substrate for 4 to 6 h. The pH optima is 5.6-6.0. The substrate concentration (IS mM) is above saturation concentration. When the logarithm of the enzyme activity was plotted against I/T (Arrhenius plot), a linear relation was obtained from IO to 37”. From the slope of this plot, the Qlo was calculated to be 1.8, and the activation energy gooo Cal/mole. DISCUSSION
In 40 serum samples, glucose was estimated both with glucose-oxidase and with o-toluidine. The differences were within 5%. This small variability is quite acceptable in view of the wide range of serum trehalase values in the sample of the normal population. The assay takes place in a very dilute serum (0.0045 ml), whereas the biogenic control tube corrects for any absorbance not related to the enzyme. It therefore Clin. Chim. Acta,
q
(1970)
349-353
VAN HANDEL
352 TABLE
III
REINVESTIGAIION OF THE SAME DONOR,
First
200
50 200
value
80
200
220
300 230
350
270
300 300 500
210
80 100
120
250 250 150 4.50 600 100 IO0
150
200
400 200
600 550 15”
180
LATER
170
400
200
MONTHS
Same donor 3-6 mods later
420 440
50 3.50 50 25
SEVERAL
160
appeared unnecessary to prepare a protein-free filtrate, even in the presence of turbidity, hemolysis or high bilirubin values. Several specimens with less than 50 trehalase units were mixed with equal parts of a standard serum of 900 units. The mixed sera always had the calculated average trehalase value, indicating that the low values were not due to inhibition of trehalase. Glucose standards added to the control tubes were always quantitatively recovered, both with the glucose oxidase and with the o-toluidine reagents. When the glucose oxidase reagent is free of trehalase, it is not necessary to use a substrate blank, because trehalose does not hydrolyze non-enzymatically. Trehalase of human serum has been recently investigated by Courtois and his students1p2. Their normal values are within the range of those in Table I. Much higher values than those reported here for the human are found in the serum of the rabbit, the squirrel, and the armadillo3. Trehalose, the only substrate known to react with trehalase, is the main blood sugar of many insects and some other invertebrates. So far, it has not been demonstrated in any vertebrate animal. The hypothesis that trehalase plays a role in the transport of glucose through the kidney is under current investigation536. ACKNOWLEDGEMENTS
Serum of blood donors was kindly provided by Dr. L. E. McHenry, director of pathology of the Brevard Hospital. The trehalase determinations were done by Mrs. Joye Chauncey. This work was supported by a grant (AI 09363) from the National Institutes of Health.
Clin. Chim. Acta, 29 (1970) 349-353
SERUM TREHALASE
353
REFERENCES I J. E. COURTOISAND J. F. DEMELIER,Bull. Sec. Chim. Biol., 48 (1966) 277. 2 J.C. GEORGET, Recherches SW la Trehalase du serum humain. Thesis, University of Paris series U-No. 446 (1966-1967).
VAN HANDEL, Cow@. Biochem. Physiol., 26 (1968) 561. 4 I.S. LUKOMSKAYA AND G. I.TARASOVA,Biokhimia, 30(1965) =JE. VAN HANDEL, .%ience, 163 (1969)1075. 6 W. GROSSMAN AND B. SACKTOR,Science, 161 (1968)571. 7 E. HULTMAN, Nature, 183 (1959)108. 3 E.
95.
Cl&z.Chim. Acta, zg (1970) 349-353