Irena Antonowicz

Developmental Pattern of Small Intestinal Enterokinase and Disaccharidase Activities in the Human Fetus

The concomitant appearance of enterokinase (EK) and trypsin activities in the human intestinal mucosa is indicative of the importance of EK as an activator of trypsinogen and therefore as the key enzyme in protein digestion. Enterokinase can be detected in fetal mucosa from the 26th week of gestation on, paralleling appearance of tryptic activity in meconium. The developmental pattern of EK activity increases with age. Between 26 to 30 weeks of gestation, the EK activity is only 6% and full term babies (40 weeks) 20% of that found in older children. In contrast, lactase studies during development show a lactase activity of only 30% in human fetuses between 26 to 34 weeks of gestation as compared to full term babies. During the same gestational period, sucrase and maltase activities reach 70% of the full term. In addition, the distributional pattern of EK differs from the disaccharidases, showing the highest activity in duodenum and the lowest in ileum, whereas disaccharidases are highest in jejunum with lower activity in duodenum and ileum. Differences in topographical distribution and time of appearance of EK and disaccharidases may be attributed to differences in orgin as well as subcellular localization of these enzymes. It is conceivable that the premature infant, between 26 to 30 weeks of gestation, is better equipped to deal with hydrolysis of alpha-glucosides than of lactose.

THE SWEAT TEST IN CYSTIC FIBROSIS

In 1953, Darling, d i Sant’Agnese, and co-workers’ opened a new area of invest igat ion when they discovered a n abnormality in function of t h e eccr ine s w e a t g l a n d s i n children with c y s t i c fibrosis. T h e s e children, when subjec ted t o a s tandard mild thermal s t r e s s for a period of one to two hours while lying quie t ly in a room maintained a t 32.2OC. and 50 per c e n t humidity, produced a s w e a t with a cons is ten t e levat ion of sodium, chloride, and potassium a s cont ras ted with control cases. Sweat from t h e abdominal a r e a w a s absorbed onto a 12 sq. cm. dry gauze pad and subsequent ly e lu ted with d is t i l l ed water for chemical ana lys i s . O n e of u s had t h e good fortune t o v i s i t Darling’s physiology laboratory i n t h e spr ing of 1954, at a t ime when d i Sant’Agnese w a s “sweat ing” two children. I w a s impressed by t h e importance of t h e work; also by t h e c o s t and upkeep of t h e experimental s e t u p a n d t h e need for well-trained technica l a s s i s t a n t s . Numerous ques t ions come t o mind when a new observat ion is made in a genet ical ly determined d i s e a s e of unknown etiology, with multiple clinical manifestations. I s h a l l l i s t a number of ques t ions but d i s c u s s on!y a few. How genera l is t h e defec t? I s i t found in all p a t i e n t s with c y s t i c f ibrosis or in t h e majority of c a s e s ? T h i s quest ion c a n only b e answered by tes t ing large numbers of pa t ien ts with a var ie ty of manifestat ions of c y s t i c f ibros is a t different ages , including infan ts with meconium ileus. Should t h e s w e a t e lec t ro ly te concentrat ions prove t o be suff ic ient ly abnormal when compared with t h e host of c l in ica l condi t ions l ikely t o b e confused with c y s t i c f ibros is and when a method c a n b e devised t o t e s t s u c h pa t ien ts a t all ages, a d iagnos t ic appl icat ion becomes possible. T h u s s u c h a t e s t could a id i n t h e detect ion of cases of c y s t i c f ibrosis in which there is no pancreat ic involvement o r only par t ia l or incomplete pancreat ic insufficiency. T h i s la t te r en t i ty did not represent c y s t i c fibrosis according t o some workers, although we gathered ev idence to indica te that i t occurred i n approximately 15 t o 20 per c e n t of our pat i en ts with c y s t i c fibrosis. In 1954 Higgins, Dooley, and Shwachman,’ a f te r consul ta t ion with Yaglou of t h e Harvard School of P u b l i c Health, developed t h e so-cal led bag method of inducing sweat , t h u s making it poss ib le t o confirm and extend t h e observa t ions of d i Sant’Agnese and h i s a s s o c i a t e s in a relat ively short time. T h e a n s w e r s t o t h e f i rs t two ques t ions appeared a t t h e end of 8 months of bag tes t ing in 239 individuals. T h e bar diagram of F I G U R E 1 i l lus t ra tes t h e e leva ted sodium v a l u e s i n 108 pa t ien ts with c y s t i c fibro-

Enterokinase and Trypsin Activities in Pancreatic Insufficiency and Diseases of the Small Intestine

The interrelationship of enterokinase and trypsin activities were investigated in 133 infants and children with a variety of gastrointestinal and pancreatic disorders. Fourteen patients with diarrhea and grade II mucosal injury revealed a significant (P less than 0.01) reduction of enterokinase, trypsin, and disaccharidase activites as compared to 59 children with normal mucosa. Nine patients with cystic fibrosis and pancreatic insufficiency had normal mucosal enterokinase activity and elevated intraluminal enterokinase activity with very low or no trypsin activity. Patients with hypoproteinemia and gastrointestinal protein loss, associated with intestinal lymphangiectasia (4 patients) and intestinal lymphoid nodular hyperplasia (3 patients), had normal or insignificant decrease of enterokinase and trypsin activities. In patients with steatorrhea, a normal sweat test, normal intestinal mucosa, and absent trypsin activity, two entities were defined. One group (3 patients) was diagnosed as Schwachman-Diamond syndrome with pancreatic insufficiency and normal mucosal and intraluminal enterokinase activity. The second group (2 patients) with absent mucosal and intraluminal enterokinase activity and normal lipase and amylase activities was diagnosed as congenital enterokinase deficiency.

Cystic fibrosis: lysosomal and mitochondrial enzyme activities of lymphoid cell lines.

Extract: Seven lysosomal enzymes and three mitochondrial enzymes were studied in cultured lymphoid cells from four patients with cystic fibrosis, three heterozygotes, one borderline patient, and six healthy controls. The lysosomes and mitochondria were isolated by differential and density gradient centrifugation. The activity of α-glucosidase, which is a lysosomal enzyme degrading glycogen, showed a marked increase in activity in the lysosomes of cultured lymphoid cells obtained from patients with cystic fibrosis as compared with the healthy controls. Two substrates were used for measuring the activity of this enzyme, the synthetic p-nitrophenyl-α-glucoside (p-NP-α-glucoside), and the physiologic substrate glycogen. The values obtained withp-NP-α-glucoside were 32.6 (SD ± 8.6) and 13.8 (SD ± 12.2) μmoles/g lysosomal protein/hr and with glycogen 543.5 (SD ± 163.3) and 188.6 (SD ± 156.3) μmoles/g lysosomal protein/hr for patients with cystic fibrosis and controls, respectively. The values of α-glucosidase in the heterozygotes were also elevated, 17.2 (sD ± 5.5), controls 13.8 (SD ± 12.2) usingp-NP-α-glucoside as substrate. When glycogen was used as substrate, the values were 226.7 (SD ± 85.3) in the heterozygote and 188.6 (SD plusmn; 156.3) μmoles/g lysosomal protein/hr in the control. The activities of the other six lysosomal enzymes—β-Dglucosidase, ‡-galactosidase, ‡-glucuronidase, N-acetyl-‡-glucosaminidase, aryl-sulfatase, and acid phosphatase—were the same in patients with cystic fibrosis as in the controls. The three mitochondrial enzymes assayed, succinic dehydrogenase, glutamic dehydrogenase, and malic dehydrogenase, showed the same activity in all subjects tested.Speculation: This study indicated that the metabolism of lymphoid cells obtained from patients with cystic fibrosis (CF) and maintained in culture for prolonged periods differs from that of cells of healthy controls. The abnormal elevated activity for α-glucosidase inlymphoid cells derived from patients with cystic fibrosis in noted when measured with synthetic substrate as well as with a physiologic substrate, glycogen. To date no defect n glycogen metabolism has been noted in cystic fibrosis although Pallvicini et al. did observe an increase in the glycogen content of cultured fibroblasts obtained from patients with cystic fibrosis. Bartman et al. found and increase in size of lysosomes in cultured fibroblasts from patients with CF by electron microscopy. These and our observations suggest the possibility of and impaired cellular metabolism in CF indicating that we may be dealing with a lysosomal disorder. Of some importance is the observation that the activity of a-glucosidase is also elevated in the heterozygote, but not to the same extent as in the homozygote. Perhaps this could offer a means of detecting the heterozygote, a possibility which should appeal to the genetic counsellor. Although numerous techniques and reports have suggested methods of identifying the heterozygote, none is practical or reliable. To be of value the test would have to take less time than required in the technique used here. For this purpose we are in the process of studying the activity of the a-glucosidase from cells derived from the patient rather than from cells grown in continuous culture.It is quite possible that patients suffering from malignant disorders or those receiving radiation or drugs may also show abnormal levels of lysosomal enzymes but may not have the same specificity; i.e., many of the lysosomal enzymes may be elevated rather than an isolated one.

Disaccharidase and Lysosomal Enzyme Activities in Amniotic Fluid, Intestinal Mucosa and Meconium

Brush border membrane bound disaccharidases (sucrase and maltase) and lysosomal enzyme (alpha-glucosidase, beta-D-fucosidase and N-acetyl-beta-glucosaminidase) activities awere studied in amniotic fluid (AF). The above enzymes except N-acetyl-beta-glucosaminidase showed a decrease in activity with gestational age beginning at about the 19th week. The activities of sucrase and maltase correlate with the morphological maturation of fetal intestinal mucosa. The distribution of disaccharidases and lysosomal alpha-glucosidase in AF and intestinal mucosa showed different patterns suggesting that these enzymes originate in diverse fetal tissues.

Studies in Meconium in Cystic Fibrosis: the Activities of alpha-D-Mannosidase, beta-Glucuronidase, beta-D-Fucosidase Acid and Alkaline Phosphatase

The specific activities of alpha-D-mannosidase, beta-glucuronidase, beta-D-fucosidase, acid and alkaline phosphatase were studied in meconium from infants with cystic fibrosis (CF) and control subjects. The study revealed significant variations in the specific activity of the enzymes except for acid phosphatase. The variations were not uniform. The activities of alpha-D-mannosidase, beta-glucuronidase and alkaline phosphatase were markedly decreased (p less than 0.001, p less than 0.002, p less than 0.001, respectively), while the activity of beta-D-fucosidase was significantly increased (p less than 0.001) in meconium from the infants with CF. It is suggested that the decreased activity of alpha-D-mannosidase and beta-glucuronidase might contribute to the accumulation of the abnormal substances in CF meconium. The highly increased activity of beta-D-fucosidase raises the possibility of an additional or alternative method for screening newborns for CF using meconium as the test material.

THIRD TRIMESTER DEVELOPMENT OF SMALL INTESTINAL ENTEROKINASE (EK) AND DISACCHARIDASE ACTIVITIES IN THE HUMAN FETUS

The early feeding of proteins and carbohydrates to premature babies necessitates knowledge of the developmental pattern of enzyme activities in the fetal gastrointestinal tract. The activities of EK and disaccharidases in small intestine obtained from 38 human fetuses between the ages of 21 and 40 weeks of gestation were investigated. EK was detected in fetal mucosa from the 26th week of gestation on, paralleling appearance of tryptic activity in meconium. The concomitant appearance of EK and trypsin activities in the human intestinal mucosa is indicative of the importance of EK as an activator of trypsinogen and therefore as the key enzyme in protein digestion. Between 26-30 weeks of gestation, the EK activity is only 6% and full term babies (40 weeks) 20% of that found in older children. In contrast lactase in 26-34 week fetuses was 30% and sucrase and maltase were 70% of the full term baby. In addition, the distributional pattern of EK differs from the disaccharidases, showing the highest activity in duodenum and the lowest in ileum, while disaccharidases are highest in jejunum with lower activity in duodenum and ileum. It is conceivable that the premature infant, between 26-30 weeks of gestation, is better equipped to deal with hydrolysis of α-glucosides than lactose, and that the very low EK activity is a major contributing factor to the very low tryptic activity.

CRITICAL AGE OF LACTASE DEFICIENCY IN CAUCASIANS

Small intestinal lactase activity in the healthy adult is either the same as in early infancy or may drop to very low levels. The incidence varies with the ethnic qroup studied but very little information is available as to the age lactase decreases. We selected 150 specimens with normal histologic appearance out of 1047 mucosal biopsies. They were derived from a heterogenous white ethnic group with failure to thrive, healthy siblings and parents, ranging in age from 6 weeks to 50 years. Mucosal lactase activity and sucrase/lactase ratios in these 150 subjects were plotted against their age. In the first 3 years of life lactase activity averaged 32.1±10.1 Mmoles/αP/min. and sucrase/lactase ratio was 1.7 ± 0.5. There was no significant difference in mean or S.D.from year to year in the first three years. After the age of 5, two separate groups emerged. One group with low lactase activity and the other with the same mean value as in the first three years. The group with low lactase included children and adults with clinical lactose intolerance and some (8) upon testing, had a flat lactose tolerance test. They consumed relatively small amounts of milk. The other qroup with high lactase activity consumed an average of one ouart of milk a day, and some (3) on testing, had a normal lactose tolerance test. This study implies that healthy Caucasian children up to age of 5 can safely consume milk without fear of lactose intolerance.

ENTEROKINASE AND INTRALUMINAL TRYPSIN ACTIVITY: DISTURBED RELATIONSHIP IN CHRONIC DIARRHEA IN CHILDREN

Enterokinase initates digestion of protein by conversion of trypsinogen into trypsin. The possible failure of this function and the interactions between the two enzymes were investigated in two groups of infants with chronic diarrhea. The first group, included six infants (age 1 to 3 months) with intractable diarrhea of infancy, revealed reduced mucosal entnrokinase activity (9.5±6.5 μM/gP/min) and low intraluminal trypsin and enterokinase activities. After treatment with I.V. alimentation the activities of enzymes returned toward normal (enterokinase values = 49.6±17.5 μM/gp/min; normal values 91.0±43.2 μM/gP/min.). The mucosal morphology of all the pretreatment biopsies showed Grade III atrophy which improved to Grade I and II after treatment. The second group consisted of eight children (age 6 months to 4 years) with chronic nonsoecific diarrhea. Their intestinal mucosa demonstrated normal morphology and disaccharidase activities but decreased activity of enterokinase (10.4±3.8 μM/gp/min.) and intraluminal trypsin. These findings suggest that enterokinase deficiency and reduced trypsin in intractable diarrhea in infancy may be a contributing factor to the protein malabsorption and consequent malnutrition. The enterokinase deficiency in chronic nonspecific diarrhea has not been reported previously and may represent a new entity.