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In vitro assessment of the intra luminal phase of gliadin digestion in coeliac disease: γ-glutamyl transpeptidase-like activity in saliva
C/it&u Chimica Aera, 130 ( 1983) 377-38 Etsevier
1
377
CCA 2515
Short
communication
In vitro assessment of the intra luminal phase of gliadin digestion in coeliac disease: ~-glutamyl transpeptidase-like activity in saliva A. George
F. Davidson
* and Michael
A. Bridges
Biochemical Diseases Clinical Service, 3. C. Children’s Hospiral, 4480 Oak Street, Vancouuer B.C., V6H 3 V4 (Canada) (Received December 30th. 1982)
Summary y-Glutamyl transpeptidase (GGTP) is an enzyme of potential relevance to the digestion of dietary gliadin, and hence of possible importance in the development of coeliac disease (CD). We have confirmed that human saliva has GGTP-like activity. Comparison of this activity between treated CD patients in long-term remission, age- and sex-matched normal control subjects, and unaffected relatives of CD patients did not show statistically significant differences. Although our results have not demonstrated a primary abnormality of this particular enzyme activity in CD, the intraluminal phase of gliadin processing is potentially important in the aetiology and pathogenesis of CD and thus deserves further study.
ln~uction Coeliac disease (CD) is one of the commonest chronic gastrointestinal diseases, affecting both children and adults. Its clinical effects vary widely but classical manifestations include diarrhoea, steatorrhoea, weight loss and malnutrition [ 11. These effects are induced in susceptible individuals by the ingestion of gluten-containing foods, whereas normal persons consuming the same diet exhibit no ill effects. While it has been shown that the toxicity of gluten-containing foods for the intestinal mucosa of persons with CD resides almost exclusively in gliadin, the alcohol-soluble fraction of gluten [2], the reason for the unique susceptibility of CD patients to this fraction is unclear. One of the major premises has been that the individual with CD lacks an enzyme normally responsible for ‘detoxifying’ gluten(gliadin) [3]. The search for this missing enzyme in CD has centered on the
* Correspondence to Dr. A.G.F. Davidson, address as above. ~9-8981/83/$03.~
0 1983 Elsevier Science Publishers B.V.
318
intestinal mucosa. Extensive study of this tissue obtained from treated and untreated CD patients over the past two decades has not been able to confirm that such a primary mucosal enzyme defect exists. Very little attention has been given to the intraluminal phase of gliadin digestion or to the role of enzymes of salivary, gastric or intestinal secretions. This is surprising; if gliadin is indeed toxic to the intestinal mucosa, it would be advantageous for the organism to ‘detoxify’ it in the gut lumen before it reaches the site of in vivo sensitivity. The intra-lu~nal phase of digestion may therefore be of vital importance to the normal ‘processing’ of ingested ghadin, and a defect in this phase might be responsible for the development of CD. In light of the possible importance of such an intraluminal ‘detoxification process’, we sought to determine whether differences could be demonstrated in secretions from the digestive tract in coeliac and non-coeliac individuals. These secretions are known to be complex mixtures of non-protein and protein material such as enzymes, many of which have not yet been adequately characterized. We chose to begin our investigations by looking for y-glutamyl transpeptidase (GGTP) in saliva. We did this because Cohen et al had suggested a role for this enzyme in the aetiology of CD [4]. Investigations by these workers and subsequent studies by others of the intestinal mucosal activity of this enzyme produced conflicting results [4,5]. However, these studies had not considered the possible presence or importance of GGTP activity within the intra-luminal phase of gliadin processing, a phase we believe may be of greater importance than the mucosal phase in the development of CD. In preliminary work, we found GGTP-like activity present in normal human saliva as had been suggested by others [6,7]. Accordingly this activity was investigated in CD and control salivary specimens, and these results are reported here. Subjects and methods The subjects selected for study included nine CD patients in long-term remission due to dietary gluten restriction, nine age- and sex-matched normal controls and five unaffected relatives of CD patients. Ages of these individuals ranged from 24-70 years. Samples of saliva were obtained after informed consent and after ascertaining that subjects had had nothing to eat, drink or smoke for at least 1 h. Salivation was induced by chewing squares of parafilm@. The first 2 ml of saliva were discarded, then the next 5 ml were collected in plastic vials. Saliva specimens were immediately aliquoted for protein and GGTP analysis. This was necessary to prevent loss of GGTP-like activity into the sediment which quickly forms in saliva upon standing. Aliquots were stored overnight at 4°C and assayed on the following day without loss of enzyme activity. The GGTP-like activity of saliva was assayed by a modification of the serum GGTP method of Naftalin et al [S]. This method was changed as follows: (1) less concentrated stock solutions of y-glutamyl-p-nitroanilide were used (6.4 mg/lO ml 0.1 mol/l glycyl glycine in 0.1 mol/l Tris-HCl, pH 7.8 buffer); this was done to prevent analytical errors we had encountered due to sedimentation of substrate from
319
the saturated solution used by Naftalin et al: (2) the incubation volume was doubied to accommodate IOO-~1aliquots of saliva; (3) the incubation time was extended from 10 min to 45 ruin without sacrificing zero order conditions; this was done to compensate for the lower enzyme activity in saliva as compared to serum; and (4) prior to spectrophotometric reading at 540 nm, tests and blanks were centrifuged at 3000 x g for 10 min to remove suspended salivary particulates which increased the background colour of the assay; this centrifugation step did not affect the concentration of chromogen in solution. Protein determinations were made by the method of Lowry et al [9]. Salivary GGTP activities were expressed both as IU/l sample fluid and as IV/g sample protein. Comparative GGTP data were analysed statistically by the Student t test for independent samples [lo].
GGTP-like activity was found in all salivary specimens assayed. This activity produced a concentration-dependent hydrolysis of the substrate y-glutamyl-pnitroanilide at pH 7.8, releasing graded amounts of the chromogen( p-nitroanaline). This activity was also found to precipitate with the mucous glycoprotein fraction of saliva, a process which could be forced to completion if salivary samples were subjected to ultracentrifugation. Table I displays the mean rt: SD data obtained for the CD and control salivary samples. Regardless of whether these GGTP activity data are expressed as IU/l fluid or as III/g protein, there does not appear to be any statistically significant difference among the three groups of subjects.
TABLE
I
y-GLUTAMYL TRANSPEPTIDASE (GGTP) TREATED CD PATIENTS IN LONG-TERM
ACTIVITY OF SALIVA SAMPLES OBTAINED REMISSION AND FROM CONTROLS
Salivary GGTP
Subject
activity ’
expressed as IU/I saliva
Statistical significance
5.58 + 2.80
Coeiiac disease (n=9) Unaffected (n=5)
expressed as IU/g salivary protein
Statisticai significance
-
2.35& 1.31
-
5.88 & 2.84
N.S.
1.8OrlrO.86
N.S.
6.OOrfr4.11
N.S.
2.19f
N.S.
b
Control (n=9)
relative of coeliac
a Meanf SD. b Statistical significance
assessed at the
FROM
1.54
p -z0.05 level by the Students t test for unpaired
samples.
a
380
Discussion Gliadins are unusual proteins, rich in glutamic acid, glutamine and proline, but deficient in basic amino acids [ 11,121. The glutamic acid residues of gliadin can form either (Y- or v-glutamyl peptide bonds [4], and these latter can be hydrolysed by y-glutamyl transpeptidase (GGTP) [4,13], an enzyme widely distributed in the gastrointestinal tract [ 141. Cohen et al [4] found depressed GGTP activity in homogenates of intestinal mucosa obtained both from treated and untreated CD patients, and, based on their results, they suggested that this reduction in GGTP in CD might represent more than a secondary effect of tissue injury. Peters et al [5], using subcellular fractionation techniques, confirmed the deficiency of brush border GGTP activity in patients with active CD, but found apparently normal levels in mucosa from CD patients in remission, thus suggesting that the decrease in mucosal GGTP was, after all, not primary. We chose to look for GGTP-like activity in saliva in CD because of the potential relevance of this activity to the intraluminal digestion of gliadin, the protein which induces intestinal mucosal damage in CD patients. Previous investigators of GGTP activity in CD had not tested for this activity in salivary or gastrointestinal secretions, nor had they considered the hypothesis that the site of abnormal gliadin metabolism in CD might reside in the intra-luminal phase of its digestion or processing. In preliminary work, we found GGTP-like activity was indeed present in human saliva, as had been suggested by others [6,7]. This work had also yielded suggestive evidence that salivary GGTP-like activity might be depressed in acute phase CD patients [15]. However, our results for both CD and control subjects overlapped widely, causing this preliminary data to be somewhat suspect. Consequently, prior to undertaking further study of CD salivary GGTP-like activity, we critically assessed our methods of handling and of assaying salivary samples. The results of this assessment were incorporated in the methodology as described in this paper. Having instituted the afore-mentioned methodological alterations, we undertook a new series of studies of GGTP-like activity in CD and control saliva. Instead of acute-phase patients, we investigated those in remission induced by dietary gluten restriction. We chose this group to ensure that our results would not be biased by secondary effects which might be seen in untreated disease. Examination of Table I shows the results of this study. No statistically significant differences in salivary GGTP-like activity were found between the CD and control groups. This suggests that salivary GGTP-like activity is not primarily affected in CD. Although this work has not demonstrated an abnormality in GGTP-like activity in salivary secretions in CD, it has provided a clear illustration of our postulate that enzymes which might be relevant to the development of CD are likely to be found in these secretions. We feel that these results reinforce our contention that more attention needs to be focused on saliva and other gastrointestinal secretions as being potentially important in the aetiology and pathogenesis of CD.
381
Acknowledgements We acknowledge the kind support of the B.C. Health Care Research Foundation and of the Vancouver Foundation. In addition, we wish to thank Prof. D.A. Applegarth, Dr. L.T. Kirby and Dr. L.T.K. Wong for their helpful advice, and Mrs. Joy Laxton for her patience and care in the preparation of this manuscript. We expressly wish to acknowledge the invaluable help of Mrs. Shirley Turtle in the performance of salivary assays. References
10 11 12 13 14 15
Falchuk ZM. Update on gluten-sensitive enteropathy. Am J Med 1979; 67: 1085-1096. Van De Kamer JH, Weijers HA. Coeliac disease: some experiments on the cause of the harmful effect of gliadin. Acta Paediatr 1955; 44: 465-469. Frazer AC, Fletcher RF, Ross CA, Shaw B, Sammons HG, Schneider R. Gluten-induced enteropathy: The effect of partially digested gfuten. Lancet 1959; ii: 252-255. Cohen MI, McNamara H, Blumenfefd D, Aries IM. The relationship between glutamyl transpeptidase and the syndrome of celiac-sprue. In: Booth CC, Dowhng RH, eds. Celiac disease. Edinburgh and London: Churchill Livingstone, 1970: 91-105. Peters TJ. Jones PE, Wells G. Analytical subcellular fractionation of jejunal biopsy specimens: enzyme activities, organelle pathology and response to gluten withdrawal in patients with coeliac disease. Clin Sci Molec Med 1978; 55: 285-292. Majewska A Pawilowska H, Potoczek A, Zaslonka B. Gamma-glutamyl transpeptidase in the saliva and dental caries (a preliminary report). Czas Stomstol 1970; 23: 675-679. Potoczek S Potoczek S, Zaslonka B, Majewska A, Pawilowska, H. Gamma-glutamyl transpeptidase in human saliva. Czas Stomatol 1970; 23: 773-778. Naftalin L, Sexton M, Whitaker JF, Tracey DV. A routine procedure for estimating serum gammaglutamy transpeptidase activity. Clin Chim Acta 1969; 26: 293-296. Lowry OL, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin reagent. J Biol Chem 1951; 193: 265-275. Mathematics and Statistics. In: Diem K. Lentner C, cds. Documenta Geigy Scientific Tables, 7th edn. Basle: J.R. Geigy, S.A., 1970. Ewart JAD. Chemistry of wheat proteins. In: Booth CC, Dowling RH, eds. Coeliac disease. Edinburgh, London: Churchill Livingstone, 1970: 1-9. Hekkens WThJM. The toxicity of gliadin: a review. In: McNicholl B, McCarthy CF, Fottrell PF, eds. Perspectives in coeliac disease. Baltimore: University Park Press, 1978: 3- 15. Meister A, Tate SS. Glutathione and related gamma-giutamyl compounds: biosynthesis and utilization. Ann Rev Biochem 1976; 45: 559-604. Naftalin L, Child VJ, Morley DA. Observations on the site of origin of serum gamma-glutamyl transpeptidase. Clin Chim Acta 1969; 26: 297-300. Davidson AGF, Bridges MA. Unpublished data. 1982.
1
377
CCA 2515
Short
communication
In vitro assessment of the intra luminal phase of gliadin digestion in coeliac disease: ~-glutamyl transpeptidase-like activity in saliva A. George
F. Davidson
* and Michael
A. Bridges
Biochemical Diseases Clinical Service, 3. C. Children’s Hospiral, 4480 Oak Street, Vancouuer B.C., V6H 3 V4 (Canada) (Received December 30th. 1982)
Summary y-Glutamyl transpeptidase (GGTP) is an enzyme of potential relevance to the digestion of dietary gliadin, and hence of possible importance in the development of coeliac disease (CD). We have confirmed that human saliva has GGTP-like activity. Comparison of this activity between treated CD patients in long-term remission, age- and sex-matched normal control subjects, and unaffected relatives of CD patients did not show statistically significant differences. Although our results have not demonstrated a primary abnormality of this particular enzyme activity in CD, the intraluminal phase of gliadin processing is potentially important in the aetiology and pathogenesis of CD and thus deserves further study.
ln~uction Coeliac disease (CD) is one of the commonest chronic gastrointestinal diseases, affecting both children and adults. Its clinical effects vary widely but classical manifestations include diarrhoea, steatorrhoea, weight loss and malnutrition [ 11. These effects are induced in susceptible individuals by the ingestion of gluten-containing foods, whereas normal persons consuming the same diet exhibit no ill effects. While it has been shown that the toxicity of gluten-containing foods for the intestinal mucosa of persons with CD resides almost exclusively in gliadin, the alcohol-soluble fraction of gluten [2], the reason for the unique susceptibility of CD patients to this fraction is unclear. One of the major premises has been that the individual with CD lacks an enzyme normally responsible for ‘detoxifying’ gluten(gliadin) [3]. The search for this missing enzyme in CD has centered on the
* Correspondence to Dr. A.G.F. Davidson, address as above. ~9-8981/83/$03.~
0 1983 Elsevier Science Publishers B.V.
318
intestinal mucosa. Extensive study of this tissue obtained from treated and untreated CD patients over the past two decades has not been able to confirm that such a primary mucosal enzyme defect exists. Very little attention has been given to the intraluminal phase of gliadin digestion or to the role of enzymes of salivary, gastric or intestinal secretions. This is surprising; if gliadin is indeed toxic to the intestinal mucosa, it would be advantageous for the organism to ‘detoxify’ it in the gut lumen before it reaches the site of in vivo sensitivity. The intra-lu~nal phase of digestion may therefore be of vital importance to the normal ‘processing’ of ingested ghadin, and a defect in this phase might be responsible for the development of CD. In light of the possible importance of such an intraluminal ‘detoxification process’, we sought to determine whether differences could be demonstrated in secretions from the digestive tract in coeliac and non-coeliac individuals. These secretions are known to be complex mixtures of non-protein and protein material such as enzymes, many of which have not yet been adequately characterized. We chose to begin our investigations by looking for y-glutamyl transpeptidase (GGTP) in saliva. We did this because Cohen et al had suggested a role for this enzyme in the aetiology of CD [4]. Investigations by these workers and subsequent studies by others of the intestinal mucosal activity of this enzyme produced conflicting results [4,5]. However, these studies had not considered the possible presence or importance of GGTP activity within the intra-luminal phase of gliadin processing, a phase we believe may be of greater importance than the mucosal phase in the development of CD. In preliminary work, we found GGTP-like activity present in normal human saliva as had been suggested by others [6,7]. Accordingly this activity was investigated in CD and control salivary specimens, and these results are reported here. Subjects and methods The subjects selected for study included nine CD patients in long-term remission due to dietary gluten restriction, nine age- and sex-matched normal controls and five unaffected relatives of CD patients. Ages of these individuals ranged from 24-70 years. Samples of saliva were obtained after informed consent and after ascertaining that subjects had had nothing to eat, drink or smoke for at least 1 h. Salivation was induced by chewing squares of parafilm@. The first 2 ml of saliva were discarded, then the next 5 ml were collected in plastic vials. Saliva specimens were immediately aliquoted for protein and GGTP analysis. This was necessary to prevent loss of GGTP-like activity into the sediment which quickly forms in saliva upon standing. Aliquots were stored overnight at 4°C and assayed on the following day without loss of enzyme activity. The GGTP-like activity of saliva was assayed by a modification of the serum GGTP method of Naftalin et al [S]. This method was changed as follows: (1) less concentrated stock solutions of y-glutamyl-p-nitroanilide were used (6.4 mg/lO ml 0.1 mol/l glycyl glycine in 0.1 mol/l Tris-HCl, pH 7.8 buffer); this was done to prevent analytical errors we had encountered due to sedimentation of substrate from
319
the saturated solution used by Naftalin et al: (2) the incubation volume was doubied to accommodate IOO-~1aliquots of saliva; (3) the incubation time was extended from 10 min to 45 ruin without sacrificing zero order conditions; this was done to compensate for the lower enzyme activity in saliva as compared to serum; and (4) prior to spectrophotometric reading at 540 nm, tests and blanks were centrifuged at 3000 x g for 10 min to remove suspended salivary particulates which increased the background colour of the assay; this centrifugation step did not affect the concentration of chromogen in solution. Protein determinations were made by the method of Lowry et al [9]. Salivary GGTP activities were expressed both as IU/l sample fluid and as IV/g sample protein. Comparative GGTP data were analysed statistically by the Student t test for independent samples [lo].
GGTP-like activity was found in all salivary specimens assayed. This activity produced a concentration-dependent hydrolysis of the substrate y-glutamyl-pnitroanilide at pH 7.8, releasing graded amounts of the chromogen( p-nitroanaline). This activity was also found to precipitate with the mucous glycoprotein fraction of saliva, a process which could be forced to completion if salivary samples were subjected to ultracentrifugation. Table I displays the mean rt: SD data obtained for the CD and control salivary samples. Regardless of whether these GGTP activity data are expressed as IU/l fluid or as III/g protein, there does not appear to be any statistically significant difference among the three groups of subjects.
TABLE
I
y-GLUTAMYL TRANSPEPTIDASE (GGTP) TREATED CD PATIENTS IN LONG-TERM
ACTIVITY OF SALIVA SAMPLES OBTAINED REMISSION AND FROM CONTROLS
Salivary GGTP
Subject
activity ’
expressed as IU/I saliva
Statistical significance
5.58 + 2.80
Coeiiac disease (n=9) Unaffected (n=5)
expressed as IU/g salivary protein
Statisticai significance
-
2.35& 1.31
-
5.88 & 2.84
N.S.
1.8OrlrO.86
N.S.
6.OOrfr4.11
N.S.
2.19f
N.S.
b
Control (n=9)
relative of coeliac
a Meanf SD. b Statistical significance
assessed at the
FROM
1.54
p -z0.05 level by the Students t test for unpaired
samples.
a
380
Discussion Gliadins are unusual proteins, rich in glutamic acid, glutamine and proline, but deficient in basic amino acids [ 11,121. The glutamic acid residues of gliadin can form either (Y- or v-glutamyl peptide bonds [4], and these latter can be hydrolysed by y-glutamyl transpeptidase (GGTP) [4,13], an enzyme widely distributed in the gastrointestinal tract [ 141. Cohen et al [4] found depressed GGTP activity in homogenates of intestinal mucosa obtained both from treated and untreated CD patients, and, based on their results, they suggested that this reduction in GGTP in CD might represent more than a secondary effect of tissue injury. Peters et al [5], using subcellular fractionation techniques, confirmed the deficiency of brush border GGTP activity in patients with active CD, but found apparently normal levels in mucosa from CD patients in remission, thus suggesting that the decrease in mucosal GGTP was, after all, not primary. We chose to look for GGTP-like activity in saliva in CD because of the potential relevance of this activity to the intraluminal digestion of gliadin, the protein which induces intestinal mucosal damage in CD patients. Previous investigators of GGTP activity in CD had not tested for this activity in salivary or gastrointestinal secretions, nor had they considered the hypothesis that the site of abnormal gliadin metabolism in CD might reside in the intra-luminal phase of its digestion or processing. In preliminary work, we found GGTP-like activity was indeed present in human saliva, as had been suggested by others [6,7]. This work had also yielded suggestive evidence that salivary GGTP-like activity might be depressed in acute phase CD patients [15]. However, our results for both CD and control subjects overlapped widely, causing this preliminary data to be somewhat suspect. Consequently, prior to undertaking further study of CD salivary GGTP-like activity, we critically assessed our methods of handling and of assaying salivary samples. The results of this assessment were incorporated in the methodology as described in this paper. Having instituted the afore-mentioned methodological alterations, we undertook a new series of studies of GGTP-like activity in CD and control saliva. Instead of acute-phase patients, we investigated those in remission induced by dietary gluten restriction. We chose this group to ensure that our results would not be biased by secondary effects which might be seen in untreated disease. Examination of Table I shows the results of this study. No statistically significant differences in salivary GGTP-like activity were found between the CD and control groups. This suggests that salivary GGTP-like activity is not primarily affected in CD. Although this work has not demonstrated an abnormality in GGTP-like activity in salivary secretions in CD, it has provided a clear illustration of our postulate that enzymes which might be relevant to the development of CD are likely to be found in these secretions. We feel that these results reinforce our contention that more attention needs to be focused on saliva and other gastrointestinal secretions as being potentially important in the aetiology and pathogenesis of CD.
381
Acknowledgements We acknowledge the kind support of the B.C. Health Care Research Foundation and of the Vancouver Foundation. In addition, we wish to thank Prof. D.A. Applegarth, Dr. L.T. Kirby and Dr. L.T.K. Wong for their helpful advice, and Mrs. Joy Laxton for her patience and care in the preparation of this manuscript. We expressly wish to acknowledge the invaluable help of Mrs. Shirley Turtle in the performance of salivary assays. References
10 11 12 13 14 15
Falchuk ZM. Update on gluten-sensitive enteropathy. Am J Med 1979; 67: 1085-1096. Van De Kamer JH, Weijers HA. Coeliac disease: some experiments on the cause of the harmful effect of gliadin. Acta Paediatr 1955; 44: 465-469. Frazer AC, Fletcher RF, Ross CA, Shaw B, Sammons HG, Schneider R. Gluten-induced enteropathy: The effect of partially digested gfuten. Lancet 1959; ii: 252-255. Cohen MI, McNamara H, Blumenfefd D, Aries IM. The relationship between glutamyl transpeptidase and the syndrome of celiac-sprue. In: Booth CC, Dowhng RH, eds. Celiac disease. Edinburgh and London: Churchill Livingstone, 1970: 91-105. Peters TJ. Jones PE, Wells G. Analytical subcellular fractionation of jejunal biopsy specimens: enzyme activities, organelle pathology and response to gluten withdrawal in patients with coeliac disease. Clin Sci Molec Med 1978; 55: 285-292. Majewska A Pawilowska H, Potoczek A, Zaslonka B. Gamma-glutamyl transpeptidase in the saliva and dental caries (a preliminary report). Czas Stomstol 1970; 23: 675-679. Potoczek S Potoczek S, Zaslonka B, Majewska A, Pawilowska, H. Gamma-glutamyl transpeptidase in human saliva. Czas Stomatol 1970; 23: 773-778. Naftalin L, Sexton M, Whitaker JF, Tracey DV. A routine procedure for estimating serum gammaglutamy transpeptidase activity. Clin Chim Acta 1969; 26: 293-296. Lowry OL, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin reagent. J Biol Chem 1951; 193: 265-275. Mathematics and Statistics. In: Diem K. Lentner C, cds. Documenta Geigy Scientific Tables, 7th edn. Basle: J.R. Geigy, S.A., 1970. Ewart JAD. Chemistry of wheat proteins. In: Booth CC, Dowling RH, eds. Coeliac disease. Edinburgh, London: Churchill Livingstone, 1970: 1-9. Hekkens WThJM. The toxicity of gliadin: a review. In: McNicholl B, McCarthy CF, Fottrell PF, eds. Perspectives in coeliac disease. Baltimore: University Park Press, 1978: 3- 15. Meister A, Tate SS. Glutathione and related gamma-giutamyl compounds: biosynthesis and utilization. Ann Rev Biochem 1976; 45: 559-604. Naftalin L, Child VJ, Morley DA. Observations on the site of origin of serum gamma-glutamyl transpeptidase. Clin Chim Acta 1969; 26: 297-300. Davidson AGF, Bridges MA. Unpublished data. 1982.