Histochemistry 51,209-218 (1977) Itistochemis T �9 by Springer-Verlag 1977

The Activity and Distribution of Gamma-Glutamyl Transpeptidase (y-GT) in Human Foetal Organs*

Norman Fleming**, Peter Groscurth, and Gonzague S. Kistler Division of Electron Microscopy, Institute of Anatomy, University of Ziirich, Gloriastral3e 19, CH-8006 Ztirich, Switzerland

Summary. The activity and distribution ofy-GT was investigated in a number of organs from human foetuses aged from 14 to 24 weeks post menstrua- tionem. Over this period, enzyme activity increased in the kidney, pancreas and thymus, but decreased in the small intestine. No trend could be estab- lished for the liver, although activity was high. In the lung, spleen, brain and adrenals, y-GT was either detectable at very low levels or could not be demonstrated. The possible relationship between y-GT activity in some human tumours and the enzyme level in the corresponding foetal organs is discussed.

Introduction

Gamma-glutamyl transpeptidase (y-GT, E.C. 2.3.2.1.) is a membrane-bound en- zyme which catalyses the transfer of the y-glutamyl moiety of glutathione and other y-glutamyl peptides to a variety of amino acids. It is believed that the enzyme participates in the y-glutamyl cycle to effect extracellular-intracellular amino acid transport (Orlowski and Meister, 1970; Meister, 1974).

The distribution and activity of y-GT have been investigated in a number of organs from various laboratory animals, including mouse, rat, rabbit and hamster (Glenner.et al., 1962; Rutenburg et al., 1969; Albert et al., 1964). Although there are large interspecific differences in the activity of y-GT in most organs, the highest level is invariably found in the kidney. The enzyme is also found in several adult human organs, where the highest level is again present in the kidney, followed by pancreas, liver, spleen, small intestine and brain. Only trace amounts are detectable in heart, skeletal muscle and lung (Albert et al., 1964; Goldberg and Martin, 1975). Serum y-GT is clinically determined as a sensitive indicator of hepatobiliary and pancreatic diseases.

* This work was supported by a grant from the ASFC, Switzerland ** Holder of a Royal Society European Science Exchange Programme Fellowship

210 N. Fleming et al.

Elevated levels are produced in inflammatory conditions and by primary liver tumours and liver metastases. Cells of these tumours and of other human malig- nancies display high levels of the enzyme. Thus, in our laboratory, high activity has been detected in bronchogenic carcinomas, which retain this activity when serially transplanted in the nude mouse. Because of the possible relationship between malignancy in adult cells and their tendency to reacquire certain foetal characteristics, we were interested to know the activity of y-GT in human foetal organs.

In an investigation of the enzyme in human foetal and adult organs, Albert et al. (1970) showed that, in the kidney, y-GT activity was higher in adults than in foetuses, whereas activity was lower in adult liver, brain and lung than in the corresponding foetal organ. The y-GT values presented by these authors for each organ were the mean of activities determined in a number of foetuses ranging in age from 5 weeks to 6 months, and no indication was given of how enzyme levels varied with different stages of foetal development. The present study was therefore undertaken to investigate the age-related pat- terns of distribution and activity of y-GT in several human foetal organs.

Materials and Methods

The 8 foetuses examined were aborted by Caesarian Section for psychiatric reasons. Foetal age (weeks post menstruationem, p.m.) was determined by crown-heel and crown-rump length (Patten, 1968), and is recorded, together with sex, in Table 1. The following organs were removed 2-4 h after abortion and examined for the distribution and/or activity of y-GT: kidney, pancreas, liver, small intestine, thymus, lung, spleen, brain, adrenal.

Histochemistry. Fragments of tissue, 1-2 mm 3, were fixed in buffered 4% paraformaldehyde (0.1 M potassium phosphate, pH 7.4) at 4 ~ for 3-5 h, with shaking. The tissues were rinsed in the same buffer for 1 h, dehydrated through an ethanol series and infiltrated with either paraffin wax (melting point 42 ~ C), or polyethylene glycol (M.W. 1000, M.P. 37 ~ C) for 2-3 h. Sections were cut at 5 gm and stained for y-GT by the method of Rutenburg et al. (1969), which utilises y-glutamyl-4-methoxy-2-naphthylamide (Vega-Fox Biochemicals, Tucson, Arizona) as substrate.

Enzyme Assay. Fresh tissue, or material which had been stored at - 7 6 ~ for up to one week, was chopped into fragments approximately 3 mm 3, blotted and weighed. The fragments were homo- genized in a pre-cooled ground glass homogenizer in 0.1% aqueous sodium cholate (w:v 1:10

Table 1. Age and sex of foetuses examined for y-GT activity.

Foetus Age (weeks p.m.) Sex

1 14 F 2 15 F 3 16-17 M 4 20-21 M 5 20-21 M 6 21-22 M 7 22 F 8 23-24 F

p .m.=pos t menstruationem, M=male , F = f e - male

Gamma-Glu tamyl Transpeptidase in H u m a n Foetal Organs 211

or 1 : 50). The homogenate was centrifuged at 25,000 g for 30 min at 4 ~ C and the clear supernatant fluid assayed for y-GT activity by the Boehringer (Mannheim) y-GT monotes t system, based on the method of Szasz (1969).

R e s u l t s

Biochemistry

An increase in y-GT activity with foetal age was found in three of the o rgans - kidney, pancreas and thymus. In the kidney (Fig. 1 a), the enzyme level remained relatively constant from weeks 14 to 20 p.m., then almost doubled between weeks 20 and 24. In both pancreas (Fig. 1 b) and thymus (Fig. 1 e) enzyme

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e Fig. 1 a-e. Variation in y-GT activity with age in five h u m a n foetal organs. Abscissa: age, weeks p.m. Ordinate: y-GT activity, m U per g wet weight. A unit (U) of enzyme activity t ransforms 1 g Mol of substrate in 1 min. at 25 ~ C

Fig, 2. Kidney of a 14 week old foetus displaying y-GT activity in the epithelium of the proximal tubules (black arrows) and of the loop of Henle (white arrows), x 210

Fig. 3. Pancreas of a 23-24 week old foetus, y-GT is localized in the exocrine part of the organ, while the islets (arrows) are negative, x 100

Gamma-Glutamyl Transpeptidase in Human Foetal Organs 213

Fig. 4. Liver of a 20-21 week old foetus. The hepatocyte surfaces forming the bile capillaries are positive for y-GT throughout the hepatic lobules, x 230

activity increased approximately threefold over the period investigated. In con- trast, y -GT in the small intestine was reduced by a factor of about nine between weeks 14 and 22 (Fig. 1 d). Enzyme levels in foetal liver displayed such variation that no pattern of either an increase or a decrease in activity with age was evident (Fig. 1 c).

Of the remaining organs, y -GT was measured at only very low levels in the lung of some specimens, and could not be detected in brain, spleen and adrenal.

Histochemistry

Kidney. In all stages of development, y -GT was detected in the cortex and medullary rays. Activity was localized in the apical region of the epithelium of the proximal tubules and of the loops of Henle (Fig. 2). In early stages of nephron differentiation, a positive reaction was noted in the epithelium of the middle limb of the "S" - shaped anlage, which portion later develops into the proximal tubule.

The glomerula and the epithelia of the distal tubules and collecting ducts were always negative.

Pancreas. In all foetuses, y -GT was confined to the exocrine component of the organ. The enzyme was localized at the luminal surface of the epithelial

Fig. 5. Small intestine of a 14 week old foetus. The epithelial cells of the villi (white arrows) and of the crypts (black arrows) exhibit y-GT activity. Note the strongly positive material (asterisks) in the lumen, x 200

Fig. 6. Small intestine of a 21-22 week old foetus, y-GT is localized at the apical surface of the villous epithelial cells (arrows). The epithelium of the crypts is negative, x 220

Gamma-Glntamyl Transpeptidase in Human Foetal Organs 215

cells lining the developing intralobular ducts and a weak positive cytoplasmic reaction was also displayed by the epithelial cells of some of the acini (Fig. 3). In the oldest foetus (23/24 weeks p.m.) enzyme activity was also demonstrated in the epithelium of the interlobular ducts.

Liver. The enzyme was invariably visualized at the hepatocyte surfaces forming the bile capillaries and this localization was consistent throughout the entire hepatic lobule (Fig. 4). No activity was found in the epithelium of the intrahe- patic bile ducts or of the gall bladder. A weak, positive reaction was observed in the cytoplasm of some haematopoietic cells in the 14 and 15 week old foetuses, whereas in older foetuses, these cells were negative.

Small Intestine. In younger foetuses (14-16 weeks), the epithelium of the villi and of the developing crypts displayed strong y-GT activity at the apical surface (Fig. 5). Amorphous material in the intervillous space was also positive for the enzyme. In the foetuses aged 20 weeks or more, activity was retained in the epithelial cells of the villi, but was not detected in the cells lining the crypts (Fig. 6),

Thymus. In all the developmental stages examined, the medulla exhibited a diffuse positive reaction which could not be assigned to any specific cell type. In foetuses older than 18 weeks, enzyme activity was also observed in the differentiating Hassall bodies.

Other Organs. The lungs of 14 and 15 week old foetuses displayed a slight reaction for y-GT in the epithelium throughout the bronchial tract, but with development (16-24 weeks), activity gradually became localized in only the most distal branches of the respiratory tree. Weak y-GT activity was also detected in the brain of all foetuses, where the enzyme was localized in the endothelium of blood vessels. The spleen of foetuses older than 20 weeks exhib- ited a positive reaction in the differentiating follicles. The adrenals were always negative for y-GT.

Discussion

In addition to the tissues studied, a number of other foetal organs, such as testis and uterus are positive for y-GT (own observation). Because of the small size of these organs in the foetus they were not considered in the present investigation.

Of the organs examined, a definite increase in y-GT activity between 14 and 24 weeks p.m. was observed in the kidney, pancreas and thymus, whereas enzyme activity in the small intestine decreased over the same period. These trends may be maintained through the remaining stages of pre- and post-natal development until y-GT reaches the level found in the mature organ. Albert et al. (1970) showed that y-GT activity in adult human kidney was 4.9 times higher than the average foetal level; but that in adult liver, brain and lung, activity was reduced by factors of 10.5, 5.4 and 5.7 respectively.

216 N. Fleming et al.

In our study, only low levels of the enzyme could be measured in the lung of some of the older foetuses, while in brain, y-GT was only demonstrated histochemically. Albert et al. (1970) did not assay the enzyme in foetal pancreas, thymus and small intestine but the same group had earlier demonstrated that activity is high in adult pancreas and relatively low in adult small intestine (Albert et al., 1964). These findings suggest that the observed pattern of develop- ment of y-GT in these two organs may continue beyond the 24th week p.m. In foetal liver, the wide range of y-GT values determined was such that no obvious trend was apparent between weeks 14 and 24 p.m. It may be that y-GT activity in this organ is particularly sensitive to extraneous factors. Greatly elevated levels of the enzyme have been detected in the liver of two foetuses aborted in cases of extrauterine pregnancy and maternal drug intoxication, while other organs in the same foetuses displayed y-GT values in the normal age-related range (own observations). Data from these foetuses were not included in the present study.

Our observations on the histochemical localization of y-GT in the organs studied are consistent with those previously reported in human foetal and adult tissues (Glenner et al., 1962; Albert et al., 1964; Albert et al., 1970), with a few exceptions. For example, although we detected enzyme activity in the Hassall bodies of the thymus medulla, we were unable to demonstrate the positive reaction described by Albert et al. (1970), as a pattern of threads arranged between the cells, and attributed by these authors to small lymphatic vessels.

In the case of the small intestine, Greenberg et al. (1967), recorded that, in adults, y-GT was localized in the apical region and in the nucleus of the villous epithelial cells, the epithelium of the crypts being negative. Our investiga- tions showed that the enzyme is present in the epithelium of both the villi and the crypts in foetuses younger than 20 weeks p.m., but persists only in the villous epithelium in older foetuses. The nuclei of epithelial cells in the foetal small intestine were always negative.

In foetal pancreas, y-GT activity was demonstrated in the epithelium of the intralobular ducts as well as in the cytoplasm of the acinar cells. In the oldest foetus (23-24 weeks p.m.), the interlobular ducts were also positive. Glenner et al. (1962) found a similar distribution of the enzyme in the adult organ.

The observed increase in enzyme activity in foetal kidneys and pancreas may simply be the result of a proportionate increase in the number of y-GT positive cells over negative cells. In the kidney, nephrons continue to be initiated up to week 32 p.m., and from this stage on, increase in kidney size is mainly due to the elongation of the enzyme-positive proximal tubules. Similarly in the pancreas, increase in y-GT activity can be related to the proliferation of the organ parenchyma relative to the stroma.

Although such differential growth patterns within the organ may account for changes in y-GT activity, at least in the case of kidney and pancreas, these variations in enzyme level may also reflect functional adaptions in cell metabolism related to the process of differentiation. In this respect, it is notable

Gamma-Glutamyl Transpeptidase in Human Foetal Organs 217

that, under certain conditions, the enzyme localization in some adult organs may revert to that of the foetal tissue. This has been demonstrated experimentally in several animal studies. Mfiller et al. (1974), showed that after portacaval shunt, y-GT in the liver of adult rats assumed the distribution normally found in foetal animals, with the corresponding increase in enzyme activity. The enzyme is also activated in adult mouse and rat liver by a range of chemical carcinogens, in which cases significantly elevated levels are detectable both in liver cells in precancerous stages, and in liver cell carcinomas (Fiala and Fiala, 1973; Kalengayi et al., 1975).

In man, a similar increase in y-GT activity has been observed in tumour- bearing livers, where the tumour cells and surrounding hepatic tissue are strongly positive. In these organs, enzyme localization is modified from the normal adult distribution to the foetal pattern; i.e. activity is no longer confined to only the periphery of the hepatic lobule (Albert et al., 1964) but becomes evident over the entire lobule (Aronsen et al., 1969; Goldberg and Martin, 1975). These changes are frequently accompanied by an elevated serum level of y-GT.

Some carcinomas of other human organs also exhibit high y-GT activity. Increased levels of the enzyme have been noted in several types of bronchial carcinoma and these levels persist in tumour fragments serially transplanted in the thymus-dysgenetic nude mouse (own observation). In renal adenocarci- nomas, the y-GT content is lower than in normal kidney tissue (Albert, 1965; own observation), and this may again represent a reacquisition of certain foetal characteristics by adult malignant cells.

In the light of these observations, a possible relationship is indicated between carcinogenesis and the ontogenic reversal of y-GT activity and distribution.

Acknowledgements. We thank Miss M. Balzer, Mrs. K. Fennel Miss R. Forster and Mr. W. Scherle for excellent technical assistance and Miss E. Meier for typing the manuscript.

References

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Albert, Z., Orlowska, J., Orlowski, M., Szewczuk, A. : Histochemical and biochemical investigations of gamma-glutamyI transpeptidase in the tissues of man and laboratory rodents. Acta histochem. (Jena) 18, 78-89 (1964)

Albert, Z., Rzucidlo, Z., Starzyk, H. : Biochemical and histochemical investigations of the gamma glutamyl transpeptidase in embryonal and adult organs of man. Acta histochem. (Jena) 37, 74-79 (1970)

Aronsen, K.F., Hfigerstrand, I., Nord~n, J.G., Pihl, B.: On the cause of the increased activity of alkaline phosphatase and gamma-glutamyl transpeptidase in serum of patients with liver metastases. Acta chir. scand. 135, 619 624 (1969)

Fiala, S., Fiala, E.S.: Activation by chemical carcinogens of y-glutamyl transpeptidase in rat and mouse liver. J. nat. Cancer Inst. 51, 151-158 (1973)

Glenner, G.G., Folk, J.E., McMillan, P.J.: HistochemicaI demonstration of a gamma-glutamyl transpeptidase-like activity. J. Histochem. Cytochem. 10, 481489 (1962)

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Greenberg, E., Wollaeger, E.E., Fleisher, G.A., Engstrom, G.W.: Demonstration of y-glutamyl transpeptidase activity in human jejunal mucosa. Clin. chim. Acta 16, 79-89 (1967)

Kalengayi, M.M.R., Ronchi, G., Desmet, V.J.: Histochemistry of gamma-glutamyl transpeptidase in rat liver during Aflatoxin B 1 - induced carcinogenesis. J. nat. Cancer Inst. 55, 579-588 (1975)

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Mfiller, E., Colombo, J.P., Peheim, E., Bircher, J. : Histochemical demonstration of y-glutamyltran- speptidase in rat liver after portacaval anastomosis. Experientia (Basel) 30, 1128-1129 (1974)

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Patten, B.M. : Human embryology, third edit. London: McGraw-Hill Book Co. 1968 Rutenburg, A.M., Kim, H., Fischbein, J.W., Hanker, J.S., Wasserkrug, H.L., Seligman, A.M.:

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Received September 10, 1976