Enzymatic, functional and ultrastructural development of the exocrine pancreas—II. The human pancreas

Comp. Biochem. Physiol., 1975, Vol. SIA, pp. 95 to 100. Pergamon Press. Printed in Great Britain

ENZYMATIC, FUNCTIONAL AND ULTRASTRUCTURAL DEVELOPMENT OF THE EXOCRINE PANCREAS-II. THE HUMAN PANCREAS N. S. TRACK, C. CREUTZFELDT AND M. BOKERMANN Division of Gastroenterology and Metabolism, Department of Medicine, University of Giittingen, D-34 Giittingen, Humboldtallee

1, Germany

(Received 18 December 1973)

Abstract-l. Enzymes and zymogen granules were detected in the pancreas from the human fetus 14.0 cm in length. 2. Enzyme (chymotrypsin, trypsin, lipase and phospholipase A) and cellular (RER profiles, Golgi apparatus, condensing vacuoles, zymogen granules) activity increased progressively until the fetus was 40-Ocm in length; hence there was a dramatic enzymatic increase until term. 3. No fetal amylase activity was detected. 4. All five adult enzyme levels were significantly elevated when compared with the fetal concentrations. 5. Enzyme activity was demonstrated in the fetal duodenal contents from 15.0 cm in length. 6. These data reveal the functional competence of the human fetal exocrine pancreas to respond to the swallowed amniotic fluid.

INTRODUCTION

MATERIALS AND METHODS

THE FETAL human pancreas contains enzymatic activity; its presence has been demonstrated by a series of studies employing mainly qualitative assay techniques (see Werner, 1948; Gschwind, 1950; Liebermann, 1966). Whether there is any relationship between this enzymatic activity and the ultrastructure of the fetal acinar cells has not been investigated. It has been proposed that amniotic fluid swallowed by the fetus plays a nutritive role during gestation (Jeffcoate & Scott, 1959; Abbas & Tovey, 1960; Bangham et al., 1961; Kerr & Keenan, 1969). For the fetus to utilize the more complex components of the swallowed amniotic fluid (protein, lipid and phospholipid) the active release of pancreatic exocrine enzymes seems mandatory. The composition of amniotic fluid changes during gestation (Cherry et al., 1965; Mandelbaum & Evans, 1969; Andrews, 1970; Bhagwanani et al., 1972; Bhagwanani, personal communication). Analogous to the adaptive nature of the adult pancreas to specific nutritional regimes, do these changes influence fetal exocrine pancreatic enzyme concentrations? To investigate these questions the enzymatic, ultrastructural and functional development of the human fetal, newborn and adult pancreatic glands has been studied. Preliminary results have been presented (Creutzfeldt et al., 1973; Track et al.,

Materials Trypsin and chymotrypsin Biochemica Test Combination -kits were obtained from Boehringer, Mannheim; neutralized. sterile olive oil from Sieafried. ZoWn; enterokinase (E.C. 3.4.4.8) from Hshst, Frankfurti M.-Hoechst; fresh eggs from hens of the Klinik’s animal house; vestopal from J. M. Jaeger, Wsenaz-Geneva; epon from Serva Feinbiochemica, Heidelberg; soluble starch (according to Zulkowsky) and all other chemicals from E. Merck, Darmstadt. Collection of human pancreatic glands, duodenal contents and amnioticfluid Pancreatic glands obtained’at abortion or operation were frozen within 30 min after delivery; those from postmortem cases after 12-36 hr. The sources of the pancreatic glands are listed in Tables 1 and 2. In eight cases from Gijttingen the duodenum was washed out twice with 05 ml saline and the two solutions were pooled and frozen at - 30°C until assay. Amniotic fluid was collected in eight cases and frozen at - 30°C until assay. Preparation ofpancreatic extracts Pancreatic extracts were prepared as previously described (Track et al., 1972). Briefly, the tissue was thawed, weighed and homogenized in 158 mM NaCl containing 20mM CaCl,. Half the homogenate was incubated with enterokinase and then both homogenate portions were centrifuged and enzymes measured in the supematants.

1973). 95

96

N. S. TRACK,

C. CREUTZFELDT AND M. BOKERMANN

Enzyme and protein determinations

Chymokypsin (mU/mg

Trypsin (E.C. 3.4.4.4), chymotrypsin (EC. 3.4.4.5), phospholipase A (E.C. 3.1.1.4), lipase (E.C. 3.1.1.3), or-amylase (E.C. 3.2.1.1) and protein were estimated as previously described (Track et al., 1972: trypsin and chymotrypsin-Biochemica Test Combination kits; phospholipase A-Figarella & Ribeiro, 1971; lipaseRick, 1969; ol-amylase-Rick & Stegbauer, 1970; protein-Stegemann, 1960).

I.0

Trypsin (mU/mg

Immunoelectrophoresis

6

Immunoelectrophoresis was performed according the technique described by Clemente et al. (1972).

protein)

to

t Tissue preparation for ultrastructural studies Pancreatic tissue was fixed and processed as previously described (Track et al., 1972). The ultrastructural preservation was not ideal due to the time elapsed between the delivery of the fetus and the removal and fixation of its pancreas. Fetal human pancreatic glands were examined from the following lengths: 8.5 cm (2), 12.5 cm (l), 14.0 cm (l), 15.0cm (2), 16eOcm (l), 17*0cm (l), 19.0 cm (l), 21-O cm (l), 23.0 cm (l), 24.0 cm (l), 26.0 cm (l), 32.0 cm (1). Ten adult samples were examined. RESULTS The length of the fetus (crown-heel) was used in this study because it is an accurate means of allotting each fetus a definite place in the gestation period.

3

~

to 20 30 N=3 8 8

40 12 8

2

I

6

Fig. 1, Mean chymotrypsin and trypsin concentrations at different developmental stages. Pancreatic homogenates ‘were incubated with enterokinase and enzyme activities measured in the supernatants. Specific activities (activity/ mg protein) were calculated for each sample separately. Means+S.E.M. were then calculated for the fetal samples in successive IO-cm growth intervals and for the newborn and adult groups.

Amylose (Ulrng protein)

Biochemical findings The levels of the five enzymes (activity/g wet weight) and protein concentrations found in the fetal, newborn and adults samples are listed in

Tables 1 and 2. The source of each sample is also listed in the Tables. The enzymes which occur as zymogens were measured after activation; hence the enzyme nomenclature. The mean enzyme levels (specific activities) from fetal samples from successive IO-cm intervals in gestation and from the newborn and adult groups are presented in Figs. 1 and 2. Very low protease levels were detected in the fetal samples from 10 to 30 cm (chymotrypsin
2d-Lioose W/ma probin)-

L

In

I-

Fig. 2. Mean or-amylase, phospholipase A and lipase concentrations at different developmental stages. Pancreatic homogenates were incubated with and without enterokinase and enzyme activities were measured in the supematants (phospholipase A with enterokinase; LXamylase and lipase without enterokinase). specisc activities (activity/mg protein) were calculated for each sample separately. Mean+ S.E.M. were then calculated for the fetal sample in successive IO-cm growth intervals and for the newborn and adult groups.

Enzymatic, functional and ultrastructural development of the exocrine pancreas--II

97

Table 1. Fetal human pancreatic enxyme concentrations Length (crown-heel) (cm) 11.5 12.5 13.5 16.5 18.5 19.5 21.5 21.5 21.5 25.0 25-O 26.5 28.0 28.0 28.0 28.0 32.5 35.0 35.5 37.0 37.0 39.5 39.5 42.0 42.0 42.0 43.0 43.0 45.0 45.0 46.0 46.0 48.0 48.0 48.0 48-O 50.0 51.0 54.0

Trypsin (mU/g)

Chymotrypsin WJh#)

Lipase (U/g)

Phospholipase A (U/g)

Protein (&ml)

Tissue SOtHoe

0 0 40 0

0 0 0 0 0 0 0

51 1 62 0 1 154 12 44

12.14 1.56 5.62 7.72 2.27 4.51 8.12 2.50 5.68 3.50 6.30 2.10 417 5.60 500 6.30 5.00 7.03 12.50 12.50 20.83 14.45 37.50 8.60 7.80 40.63 11.25 18.75 41.25 48.44 17.50 89.58 12.80 lO*OO 18.75 35.94 62.50 I115.63 57.50

22.70 29.50 46.88 31.50 3400 53.57 26.10 25.00 30.51 5446 30.95 27.62 54.91 29.05 25.24 33.33 25.71 6390 54.63 27.62 64.88 48.75 42.38 25.24 28.10 57.56 22.86 40.50 87.81 56.10 64.88

LON GOP LON GOP GOP LON LON GOP LON LON LON LON LON LON LON LON LON GPT LON GPT GPT LON LON GPT LON LON GPT GPT GPT GPT LON GPT

30.95

GPT

46.19 52.23 55.36 62.19 75.12 69.68

GPT LON LON LON GPI GPT

3 12 1: 91: 19 0 6 3 8 7 z 26 49 30 2 57 198 23 105 64

3 0 5 0 8 0 0 0 0 0 0

4

119 869

73 44 114 806 241

167 136 1;; 57 116 150 254

0 0

1219 131

60

55 181 53 913

96 96

31 18 275 3 40 4 69 44 123 0

11: 235 104 184 513 383 1425 392 88 575 164 2375 3375 1532

51.79

Trypsin, chymotrypsin, or-amylase,lipase, phospholipase A and protein were determined as outlined in the text. No a-amylase activity was detected. The enzyme concentrations are expressed as activity/g wet wt. and the protein as mg/ml. The values presented represent single determinations. The reproducibility of the enzyme estimations was in the range of + 5 per cent. The tissue sources were LONLondon, GOP-Giittingen operation and GPT-Giittingen pathology. values from the 3-day-old samples (15.0 U/mg) were in the same range as the largest fetuses. The 3-weekold and adult values were markedly higher (100-130 U/mg). No ar-amylase, lipase nor phospholipase A activities were detected in the amniotic fluid. The lipase activity detected in the duodenal contents of eight fetuses is depicted in Fig. 3. Over the portion of the gestation period studied, an increase from 0.04 to 5.00 U/ml was observed. During pancreatic function tests in adults, lipase levels in the duodenal contents ranged from 100 to 260 U/ml for basal secretion and from 344 to 1430 U/ml after CCK stimulation (Fig. 3). 4

The results from an immunoelectrophoresis are depicted in Fig. 4. The control pancreatic juice (top) gave rise to a large number of strongly staining precipitation arcs. The duodenal contents from a 23-O-cm fetus (bottom) gave rise to one strongly staining, two medium staining and three weakly staining precipitation arcs. The three heavier arcs were tentatively identified as lipase, trypsin 1 and anionic chymotrypsin. No precipitation arcs were found in all smaller fetal samples tested (10.0, 15.0, 17.0, 18.0 cm). Also, no discernible arcs were apparent when amniotic fluid (neat and ten times concentrated) was examined.

N. S. TRACK,C. CREUTZPELDT ANDM. BOKE~MANN

98

Table 2. Newborn and adult human pancreatic enzyme concentrations Trypsin (mu/g)

Age 3 days 3 days 3 weeks 43 years 24 years 50 years 38years 38years 24years

Chymotrypsin (mU/g)

Amylase (v/g)

Lipase (U/g)

Phospholipase A (v/g)

Protein @g/ml)

Tissue source LON LON GPT GOP GOP GOP GOP GOP GOP

1309

145

0

444

22.50

5268

2145 11005 9775 10811 7282 4852 10145 -

92 273 596 453 278 169 1362 168

0 0 2943 6287 4809 2039 5461 3115

525 3783 19163 4817 8067 4604 18748 13977

43.75 11.25 246.25 475.13 205.34 77.02 250.18 263.43

65.18 E! 93.82 73,20 52.80 98.21 84.20

Trypsin, chymotrypsin, or-amylase, lipase, phospholipase A and protein were determined as outlined in the text. The enzyme concentrations are expressed as activity/g wet wt and the protein as mg/ml. The vahres presented represent single determinations. The reproducibility of the enzyme estimations was in the range of +_5 per cent.

.&

*.

IO Fetal,

20

cm

Bz

s

Cx%&ulated Adult

Fig. 3. Lipolytic activity in the fetal and adult duodenal contents. The fetal duodena were washed out with saline and the lipase activity estimated in the solution. For the adult levels, basal and CCK-stimulated samples were aspirated from the duodenum and their lipase contents were determined.

Ultrastructural findings

Pancreatic cells from 8*5-cm fetuses were in a proto-differentiated state, consisting mainly of undifferentiated tubular cells. No zymogen granules were seen (Fig. 5). There were prominent junctional complexes and few free ribosomes present. The electron lucent lumen was fringed by microvilli with some extruding cilia (Fig. 5). Microtubules and glycogen were also present. An increase in free ribosomes was found in the 12*5-cm sample. The cells contained few short cistemae of rough endoplasmic reticulum (RER) often found in close proximity to mitochondria (Fig. 6). The pancreas

from the 14.0-cm fetus displayed few cells with organized RER. The cells were characterized by active Golgi zones, an increase in mitochondria, small zymogen granules and some lysosomal granules. Enhanced activity of the RER, dilated Golgi zones, condensing vacuoles and some zymogen granules of varying size and shape were found in the pancreatic cells of 15%crn fetuses. The lumen frequently contained some electron opaque material (Fig. 7). During the 15+)-19-O cm growth interval a gradual increase of cellular activity and zymogen granule content was noted. The granules were pale and of varied size and shape. Many undifferentiated tubular cells were still present. During the 21-O-26.0 cm growth interval, a greater number of pancreatic cells displayed a definite increase of both cellular activity and zymogen granule content (Fig. 8). The zymogen granules appeared to be paler than in the adult pancreas. During the 150-32.0 cm growth interval so-called “mixed exocrineendocrine” cells were observed (Fig. 9). In the 32O-cm fetus even more pancreatic exocrine cells showed highly organized, even lamellar RER. The number of zymogen granules increased and most possessed a greater electron density; some non-spherical granules were still observed. Condensing vacuoles, many containing flocculent material (Fig. lo), and prominent Golgi zones were pronounced features of these cells. Most ductular lumina contained some electron opaque material (Fig. 11). The adult exocrine pancreatic cell contained a prominent granular endoplasmic reticulum. The convoluted interconnected cistemae of different circumferences usually contained electron opaque material. The Golgi apparatus was prominent and in apposition to the cistemae of the RER. Condensing vesicles were located close to the Golgi apparatus. Abundant dense, mature zymogen granules were seen in the ceil apex surrounded by a tightly fitting limiting membrane (Fig. 12). Centro-acinar cells

Fig. 4. Immunoelectrophoresis of duodenal contents of a 23.0-cm fetus. Adult duodenal juice was run as a control (top) giving rise to a large number of strongly staining precipitation arcs. In the fetal sample (bottom), hpase (I-), trypsin I (T) and anionic chymotrypsin (C) were detected as well as three other faintly staining precipitation arcs.

Fig. 5. Pancreatic duct cells of 8.5 cm fetus. Electron translucent lumen fringed by microvilli with protruding cilium. Undifferentiated cytoplasm, some microtubules and junctional complexes. No zymogen granules. Screen magnification (SM.) SM. x 8000. Fig. 6. Two mitochondria

in close apposition to cisternal profiles of the RER in a pancreatic acinar cell of a 12.5-cm fetus. SM. x 27,000.

Fig. 7. Ten portions of pancreatic acinar cells of a 150 cm fetus. Zymogen granules are pale and of varied size and shape. Single short profiles of RER. Lumen fringed by microvilli contains electron opaque material. S.M. x 8000. Fig. 8. Four portions of pancreatic acinar cells of a 21.5~cm fetus. Prominent Golgi area, lamehated RER and condensing vacuoles of different electron density. S.M. x 8000.

Fig. 9. “Mixed endocrine-exocrine” cell in the pancreas of a 21.0-cm fetus. Typical alpha and zymogen granules appear to be in the same cell. SM. x 8000. Fig. 10. Pancreatic

acinar cell of a 32.0-cm fetus. Zymogen granules and condensing vacuoles of Some contain “flocculent” inclusions. RER is well developed. S.M. x 8000.

varied size, shape and electron density.

Fig.

11. Apical portions

of pancreatic acinar cells from a 32.0-cm fetus. The lumen, by microvilli, contains electron opaque material. S.M. x 8000.

fringed

partially

Fig. 12. Two adult pancreatic acinar cells containing an abundance of dense zymogen granules and some prominent cisternae of RER. Between these two cells is a centro-acinar cell characterized by the absence of secretory granules and a structurally unspecialized cytoplasm. The apical surfaces of all three cells have microvilli projecting into the lumen. S.M. x 8000.

Enzymatic, functional and ultrastructural development of the exocrine pancreas-II were also observed. Mixed cells were rarely encountered.

endocrinsexocrine

DISCUSSION During progressive stages of gestation, the fetal human pancreas contains higher enzyme activities and the pancreatic acinar cells show signs of enhanced ultrastructural organization and activity. Results from the present study (Fig. 1) confirm the increase of proteolytic activity found by Werner (1948) and show that the lipase and phospholipase A activities (Fig. 2) also exhibit an augmentation during the latter part of gestation. In studies with human material one must always consider the problem of tissue preservation. Pancreatic tissue characterized by a degenerate macroscopic and/or microscopic appearance was eliminated from this study. Since the same criteria were employed for all pancreatic tissue, it seems unlikely that the higher enzyme levels detected in the latter stage of gestation are attributable to the state of tissue preservation. Concomitant increases of substrate (Bhagwanani et aI., 1972; Bhagwanani, personal communication) and enzymes (Fig. 2) were found for lipase and phospholipase A during the latter stage of gestation. During this same stage of gestation, when there is a decrease in amniotic fluid protein concentration (Cherry et al., 1965; Mandelbaum & Evans, 1969; Andrews, 1970), the pancreatic proteases are increasing (Fig. 1). These seemingly contradictory patterns are resolved by the fact that the fetus swallows more amniotic fluid and retains more amniotic fluid protein necessary for growth during this period (Mandelbaum & Evans, 1969). No amylase activity was detected in the fetal and newborn extracts (Fig. 2). Amniotic fluid does not contain starch. Thus, these results demonstrate that the human fetal pancreas has the capacity to adapt its enzyme biosynthesis to changes in amniotic fluid, the nutritional environment. This same enzyme adaption principle explains the enhanced enzyme activities discovered in the newborn and adult extracts (Figs. 1 and 2). Detection of significant lipolytic activity in the duodenal contents of human fetuses commences at a length of 20.0 cm (Fig. 3). These “stimulated” fetal values are at least twenty times lower than adult basal levels. The immuno-electrophoresis confirmed the presence of lipase activity in the duodenal contents of a 23*0-cm fetus and illustrated the presence of other enzymes (Fig. 4). The absence of any detectable enzymatic activity in the amniotic fluid proves that the pancreatic enzymes found in the fetus are not of maternal origin. This discovery of enzymatic activities in the duodenal contents of 20.0 cm and larger fetuses reveals the functional importance of the fetal pancreas during this and later stages of gestation. Furthermore, it demonstrates that the pancreas of a premature infant does have

99

the enzymatic potential to digest unsplit protein food (Madley & Dancis, 1949). Ultrastructurally, the pancreatic glands of human fetuses of 8.5 cm length were comprised of undifferentiated tubular cells containing no zymogen granules. Zymogen granules were first detected at 14.0 cm length. Enzyme activity also was detectable at this stage. Like & Orci (1971) and Wellmann et al. (1971) have reported similar ultrastructural findings concerning the initial appearance of zymogen granules. The zymogen granules in each acinar cell increased in number and electron density as the fetus developed towards term providing ultrastructural evidence for the enhanced enzymatic activity detected during the latter part of gestation (Figs. 1 and 2). Microtubules are present in the pancreatic glands of the smallest fetuses examined. Their presence in the absence of zymogen granules suggests that at least in these undifferentiated acinar cells they may play a role in maintaining cell shape by imparting stiffness to certain areas (Fawcett, 1969). Human fetal pancreatic cells classified as mixed cells were found in fetuses from 15.0 to 32.0 cm long. Since acini and islets have a common ductular origin, embryologically, these cells may be interpreted as being a stage in development at which the final identity of the cell is not as yet determined. Mixed cells were found rarely in the adult human pancreas. Evidence for the existence of mixed or intermediate cells in the normal adult (amphibian, avian and mammalian) pancreas has been presented (see Mehned et al., 1972); Melmed et al. suggest that these intermediate cells may be a response to a metabolic demand (1973) or may be a distinct ab initio category of cells (1972). However, one must always consider the possibility that these cells arise from an imperfect state of preservation and/or a fixation artefact. Like & Orci (1971) failed to identify any mixed endocrineexocrine cells in twenty human fetuses. Frexinos et al. (1973) examined tissue fragments from twelve subjects with chronic pancreatitis during surgical removal of the pancreas and found ultrastructural evidence for “acinar-islet” cells. They believe that the so-called acinar-islet cells may occur from a scrambling of cells after a breakdown of membranes. Munger (1973), after investigating piscine, avian and mammalian pancreatic glands, stated that “acinarislet transformation is an artefact of cytoplasmic soup”. This study shows the close correlation between the enzymatic and ultrastructural events during pancreatic exocrine development in the human fetus. The detection of enzyme activities in the duodenal contents demonstrates that the human fetus has the capacity to release enzymes in order to utilize the swallowed amniotic fluid for nutritive purposes. would like to thank Dr. G. A. (Pediatric Research Unit, Guy’s Hospital,

Acknowledgements-We

Machin

100

N. S. TRACK, C. CR&JT~T

London) and Dr. U. Junge (Department of Medicine, University of Gbttingen) for their assistance in the collection of the fetal pancreatic glands. We are most grateful to Dr. C. Figarella for supplying us with antibody against human pancreatic juice and to her and Dr. de Care for their guidance in the immunoelectrophoresis technique. We thank Miss H. Dijrler and Mrs. H. Uhde for their conscientious assistance with the ultrastructural studies. REFERENCES ABBAs T. M. & TOREYJ. E. (1960) Proteins of the liquor amnii. Br. med. J. 1,4X-419. ANDES B. F. (1970) Amniotic fluid studies to determine maturity. Pediat. Clin. N. America (Philad.) 17, 49-61. BANGHAM D. R., HOBBSK. R. & TEE D. E. H. (1961) The origin and nature of the liquor amnii in the rhesus monkey; a new protein with some unusual properties. J. Physiol., Land. 158, 207-218. BHAGWANANI S. G., FAHMYD. & TURNBULLA. C. (1972) Prediction of neonatal respiratory distress by estimation of amniotic fluid lecithin. Lancet i. 159-162. CHERRYS. H., K~C!HY?AS. & ROSENFIELDR. E. (1965) Bilirubinprotein ratio in amniotic fluid as an index of the severity of erythroblastosis fetalis. Obstet. Gynecol. 26,826832. CLEMENTEF., DE C&o A. & FIGA~ELLA C. (1972) Composition du sac pancreatique humain. Etude immunoenzymologique. Europ. J. Biochem. 3, 186 193. CREUT~FE~T C., TRACK N. S. & B~KERMANNM. (1973) Enzymatic and ultrastructural development of the fetal human and bovine exocrine pancreas. Communication at the VIth Symposium of the European Pancreatic Club, Goteborg, Sweden, 24-26 May, 1973. Abstract 13. FAWCETT D. W. (1969) The Cell, An Atlas of Fine Structure. Saunders, Philadelphia. FIGAR~LLAC. & RIBEIROT. (1971) The assay of human pancreatic phospholipase A in pancreatic juice and duodenal contents. Scand. J. Gastroent. 6, 133-137. Farxr~os J., BUGAT R., BA~TIEM. J. & RIBET A. (1973) The problem of the so-called “acinar-islet” cells in human chronic pancreatitis. Communication at the VIth Symposium of the European Pancreatic Club, Goteborg, Sweden, 24-26 May, 1973. Abstract 82. GSCHWMD R. (1950) Das Verhalten der Panlcreasenzyme bei Frtihgeburten~ Ann. Paediat. (Basel) 175, 169-184. JEFFCOATET. N. A. & Scorr J. S. (1959) Polvhvdramnios and oligohydramnios. Can. ‘Med: Assoc.-J. 80, 77-86. KERR G. R. & KENNAN A. L. (1969) The free amino acids of amniotic fluid during pregnancy of the rhesus monkey. Am. J. Obstet. Gynecol. 105, 363-367.

AND M, B~KERMANN J. (1966) Proteolytic enzyme activity in fetal pancreas and meconium, Gastroenterology 50, 183190. LIKE A. & ORCI L. (1971) Embryogenesis of the human pancreatic islets : a light and electronmicroscopic study. Diabetes 21, Suppl. 2, 51 l-534. MADLEXS. & DANCI~J. (1949) Proteolytic enzymes of the premature infant. Pediat. (Springfield) 4, 177-182. MANDELBAUMN. & EVANS T. N. (1969) Life in the amniotic fluid. Amer. J. Obstet. Gynecol. 104, 365377. MELMED R. N., BENITEZ C. J. & HOLT S. J. (1972) Intermediate cells of the pancreas-I. Ultrastructural J. Cell Sci. l&449475. characterization. MELMED R. N., T’URNERR. C. & HOLT S. J. (1973) Intermediate cells of the pancreas-II. The effects of dietary soybean trypsin inhibitor on acinar-B cell structure and function in the rat. J. Cell Sci. 13, 279295. MUNGER B. L. (1973) Communication in the workshop on “Cytological composition of pancreatic islets” at the VIIIth International Diabetes Federation Congress, Brussels, Belgium, 15-20 July, 1973. RICK W. (1969) Kinetischer Test zur Bestimmung der Serumlipaseaktivitlt. Z. klin. Chem. klin. Biochem. 7, 530-539. RICK W. & STEGBAUER H. P. (1970) a-Amylase. Messung der reduzierenden Gruppen. In Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.), Band I, pp. 843-853. Verlag Chemie, Weinheim. STEGEMANNH. (1960) Formarm ‘dolyse von Proteinen. Die Einwirkung von Formamid auf Gelatine. HoppeSeyler’s Z. physiol. Chem. 319, 64-86. TRACK N. S., BCXERMANNM. & CREUTZFELDT C. (1973) Ultrastructural, enzymatic and functional studies of the fetal human exocrine pancreas in relation to the amniotic fluid. Communication at the IVth Meeting of the Diabetes Pregnancy Group of the European Association for the Study of Diabetes, Brugge, Belgium, 12-14 July, 1973. TRACKN. S., B~KERMANNN., CREUTZFELDT C., SCHMIDT H. & CREUTZFELDTW. (1972) Enzymatic and ultrastructural development of the bovine exocrine pancreas. Comp. Biochem. Physiol. 43B, 313-322. WELLMANNK. F., VOLK B. W. & BRANCATOP. (1971) Ultrastructural and insulin content of the endocrine pancreas in the human fetus. Lab. invest. 26, 97-103. WERNER B. (1948) Peptic and tryptic capacity of the digestive glands in newborns. Acta paed. (Uppsala) 35, Suppl. 6,49-80.

LIEBERMAN

Key Word Index-Human pancreatic enzymes; human exocrine pancreas development; ultrastructure of human fetal exocrine pancreas; nutritive role of human amniotic fluid; release of fetal exocrine enzymes.