The control group was composed of nine infants with normal weight/height z scores and normal mucosal histology, and were thus viewed as normal subjects (N) within our experimental design. The jejunal tissue for this group was taken from nine Brazilian patients admitted to the hospital for the Kasai surgical procedure for biliary atresia. The specimens were obtained during the routine operative procedure. The N subjects were younger and of different sex ratios than the malnourished infants, but they were matched for height and weight, and the z score and histologic criteria for their admission to the study were insensitive to age and sex ratio differences. As a confirmation, we tested older control subjects (8–10 years of age) without biliary atresia who were selected by these same criteria, and could detect no effect of age, biliary status, or biopsy procedure on the control mucosal histology and maltase activity presented in this report (data not shown). Jejunal tissue from a 4-year-old organ donor (donor IV) was used for making monoclonal antibody and for internal standards throughout the assays.
The 24 M and 9 N subjects (Table 1) were admitted to the Instituto da Crianças, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (ICR:HC FMUSP), Brazil. The organ donor tissue was collected at one of the Baylor College of Medicine–affiliated hospitals, The Methodist Hospital of Houston, Texas, U.S.A. The investigation had been approved by the Human Investigation Review Boards of the Baylor College of Medicine and affiliated hospitals and of the Department of Pediatrics ICR:HC FMUSP, and signed parental consent was obtained for each subject. Clinical examinations and data were recorded using the procedures and data management system reported previously (1,24).
Biopsy tissues were obtained from the M children after a 2-to 3-week hospital stay. These are the same biopsies reported in our previous publication (1). The jejunal tissues were obtained using a multiport pediatric Crosby–Kugler capsule, as previously described (1). Each specimen was divided as follows: a portion in Zenker's solution, embedded in paraffin, sectioned, and stained for light microscopy and diagnostic histology; and a portion embedded in an optimal cutting temperature embedding compound (OCT, Tissue-Tek, Elkhart, IN, U.S.A.), frozen in liquid nitrogen, and used for immunofluorescence, enzyme histochemistry, and RNA isolation. The remainder was frozen at −70°C until used for biochemical assay, at which time the tissue was thawed and homogenized in Dulbecco's phosphate-buffered saline containing protease inhibitors (1). In the N children undergoing the Kasai procedure for biliary atresia, 1 to 2 cm of the jejunum immediately distal to the ligament of Treitz was obtained by surgical resection during the construction of the Roux-en-Y anastomosis. Tissue was treated as described.
The results of the maltase assays were not reported in our previous publication (1). Briefly, maltase activity was determined in biopsy homogenates using 0.112 M maltose as the substrate (1). Glucose production was measured on a an autoanalyzer (COBAS Fara II; Roche Diagnostic Systems, Montclair, NJ, U.S.A.), using a glucose oxidase kit (Glucose Trinder 100; Sigma Chemical Co., St. Louis, MO, U.S.A.), and Monitrol (Roche Diagnostic Systems) as the glucose control (1). Protein was measured using a bicinchoninic acid protein assay kit (Pierce Co., Rockford, IL, U.S.A.). Activity was expressed as units (U = μM/min)/g protein (U/g protein). Normal values were those from our laboratory, and hypomaltasia was defined as less than 93 U/g protein (16).
Villous atrophy was judged from the hematoxylin and eosin–stained, paraffin-embedded or frozen sections and graded 1 to 4+ by a single histologist who graded the slides in blinded fashion (1). The mean histology grades were the same as in our previous report (1). Absence of atrophy (1+) was estimated by comparison with tissue from an organ donor, and 4+ was judged in reference to children with active celiac disease (1). For purposes of comparison, in the context of other M values, villous atrophy was recalculated as a percentage of N villous length by taking the reciprocal of the grade of atrophy (in this case, normal = grade 4 in Table 4).
Fresh assays of MGA, villin, and β-actin mRNA were performed in a subset of 10 M and 9 N subjects from Table 1 for this investigation. The subset comprising Table 3 was defined by adequacy and quality of biopsy RNA for the assay of a housekeeping gene message, β-actin. The assays for SGLT 1 were performed at the time of our previous investigation but were not reported in our previous article, which reported only LPH and SI messages (1). The present investigation used the same small intestinal RNA as our previous publication (1). Fresh reverse transcription–polymerase chain reaction (RT-PCR) analyses of mRNA were performed according to our published procedures (1). Briefly, five frozen tissue sections (10 μm) were cut from the OCT block at −20°C and attached to glass slides. RNA was isolated from scraped sections (RNeasy kit; Qiagen, Chatsworth, CA, U.S.A.). The total RNA was quantitated by optical density (OD) measurement at 260 nm. Fresh reverse transcription was performed using 100 ng of RNA at 37°C for 2 hours. The 50-μl reaction volume was composed of 10× buffer, 4 mM deoxyribonucleoside triphosphate (dNTP), 4 U RNase inhibitor, 30 ng random nonamer primers, 20 U M-MLV reverse transcriptase, and diethyl pyrocarbonate (DEPC)-treated water (RT-PCR Kit; Stratagene, La Jolla, CA, U.S.A.). The RT reaction product was amplified by PCR (Stratagene). The amplification primers for human MGA, SI, and SGLT 1 were synthesized (Genosys, The Woodlands, TX, U.S.A.). Maltase primers 5` (1669–1690) and 3` (2049–2028) were based on our cloning primers (2). SGLT 1 primers were those described by Wang et al. (25) Villin primers were those described by Fajardo et al., (26) and primers for human β-actin were purchased (#5211.h; Continental Laboratory Products, San Diego, CA, U.S.A.). The PCR reactions had volumes of 100 μl including 10× buffer, 0.8 mM dNTP, the specific primers (0.4 μM MGA, SGLT-1, or villin, and 0.5 μM total β-actin), 2.5 U Taq polymerase, and 3 μl of the RT reaction product. After a hot start at 94°C for 5 minutes, the reaction was stepped through 30 cycles of 52° for 40 seconds, 70° for 60 seconds and 94°C for 30 seconds in a thermal cycler (model 480; Perkin-Elmer, Foster City, CA, U.S.A.). SGLT 1 reactions were taken to 35 cycles because of low copy numbers. A 5-μl sample of each PCR product was visualized on a 1.5% agarose gel run in TAE buffer containing 0.5 μg/ml ethidium bromide. To quantitate the amplimers, 10 μl of the RT-PCR reaction mixture was assayed as previously described by liquid chromatography (LC) with the DEAE-NPR column (TSK kit; Perkin-Elmer) (1). RNA from organ donor IV was run in parallel with the experimental subjects as an internal standard for the assays. The LC results were expressed as nanograms amplimer/100 ng total tissue RNA. The amplifications from diluted donor IV total intestinal RNA were linear (r = 0.995–0.999) in the calibration range of 10 to 180 ng. Within-run LC coefficients of variation were less than 10%. The specificity of all PCR amplimers was confirmed by sequencing with an automated sequencer (model 373A; Applied Biosystems, Foster City, CA, U.S.A.). The quality of mRNA was confirmed by parallel amplifications from the same cDNA with the β-actin primers.
Subject data were maintained using the SIR Database Management System (SIR, Inc., Evanston, IL, U.S.A.) (1). Statistical analyses were performed using the Minitab programs (Minitab, Inc., State College, PA). Categorical variables were analyzed with χ2 or Fisher's exact test. All t-tests were computed using Bonferroni's correction. Numeric variables were analyzed by one-way analysis of variance (ANOVA). Missing data were excluded from consideration and account for the variations of sample size (Tables 1–4). Only complete data sets were used for regression analyses.
Evaluation of the nutritional status, as indicated by age-independent measures of growth and body composition, revealed severe malnutrition characterized by growth failure and tissue wasting in the M group. The nutritional status (weight/height z scores) of the infants in the M group differed significantly (P = 0.003) from that of the N subjects (Table 1). The M group had significant (P = 0.006) morphologic alterations of the jejunum with partial villous atrophy (Table 1). A correlation between villous atrophy and weight/age z score was present (r = 0.65, n = 22).
Reductions in the enzyme activities of maltase were present in the M subjects. Mean enzyme activity (units per gram of biopsy sample protein) for the nutritional groups is shown in Table 2. Maltase activities were reduced in the M specimens (to 37% of N) but statistical significance was marginal (P ≈ 0.15). Maltase activity was below normal (<93 U/g protein) in 13 of 25 M subjects. Maltase activities were within the normal range in all the N subjects. A decline in activity as weight declined from standard for age (z score) was not found for maltase activity.
The mRNA for maltase, SGLT 1, villin, and β-actin was assayed by quantitative LC amplimer measurement. Except for SGLT 1, all were amplified under identical conditions from freshly transcribed total RNA. SGLT 1 message quantitation was performed at the time of our previous study of lactase expression in the same subjects (1). Inclusion of M and N subjects in the subgroups reported in this and our previous publication (1) was based on adequacy of β-actin amplification from the biopsy specimen RNA. The present results are summarized in Table 3.
Mean maltase activity was 83 ± 52 and 224 ± 78 U/g protein in the M and the N subgroups, respectively (P = 0.001). The mean quantity of MGA mRNA amplimers was 218 ± 167 ng/100 ng RNA in the M and 485 ± 207 ng/100 ng in the N subgroups, respectively (P = 0.016). (The N values differ slightly from those previously reported  because one subject was discovered to have adult-type hypolactasia. The RT-PCR for this subject were not included in the present investigation.) SGLT 1 amplimers were 5585 ± 1936 and 8389 ± 3193 ng/100 ng RNA (P = 0.057) in the M and N groups. Villin amplimers were measured at 1179 ± 508 ng in M and 2245 ± 607 ng in N (P = 0.003). The mean mRNA amplimers for β-actin were 4180 ± 758 ng and 4794 ± 957 ng in the M and N subgroups, respectively, and were not significantly different (P = 0.185). The quantity of MGA amplimer correlated with maltase activity (r = 0.32, n = 15). There was a correlation between MGA and SGLT 1 mRNA (P = 0.67, n = 15).
Villin mRNA concentration correlated with histologic grade of villous atrophy (r = 0.69), and villin/β-actin ratios correlated with villous atrophy (r = 0.76). SGLT 1 and SGLT 1/β-actin ratios also correlated with grade of atrophy (r = 0.47 and 0.54, respectively). Maltase mRNA correlated with villous atrophy (r = 0.73), but β-actin did not (r = 0.01, n = 15).
MGA and SGLT 1 messages correlated with villin message (Fig. 1). The regression equations are shown in the legend to Figure 1 (r ≥ 0.74). The intercepts ± SD were near zero for MGA and above zero for SGLT 1. These intercept values are consistent with the M values normalized to villin message in Table 3.
Levels of maltase enzyme activity were reduced to approximately half of normal in our malnourished Brazilian infants. The loss of enzyme activity was similar to that reported in malnourished infants in other countries (27–47). The degree of villous atrophy was comparable to that reported in malnourished adults as well as children from other countries (48–67). Maltase activity levels also have been reported to be reduced in children and adults with other atrophic mucosal disorders such as intractable diarrhea of infancy and celiac syndrome (21,22,69–76). Although the association of villous atrophy has been assumed, there has been no direct proof of this association. In the present investigation, the mechanism for the reduction of mucosal maltase was examined in the context of villous atrophy, other brush border carbohydrate hydrolases, and the glucose transporter.
In all the intestinal mucosal disorders associated with villous atrophy, maltase activity has always correlated with sucrase activity (21,22,69–76). It has been reported that 80% of the maltase activity in human small intestine is contributed by SI, with the remaining by MGA (5). This correlation of maltase activity with sucrase activity was confirmed in the present study (r = 0.95, n = 32). This correlation has been attributed to the overlap of the two enzyme specificities for the maltose substrate used in the assays (21,69–76). In the present study we demonstrate, for the first time, that the correlations between the two enzyme activities exist at the mRNA levels (r = 0.73, n = 16) and cannot be explained by the overlap of enzyme specificity for maltose substrate (5).
We previously suspected that sucrase activity in malnourished infants was due to the loss of enterocytes secondary to villous atrophy and deduced that lactase message was downregulated (1). We also demonstrated that our malnourished infants with hypolactasia did not have the heterozygotic allelic polymorphism that is characteristic of all reported hypolactasic adults (1). To test the hypothesis that maltase was reduced by loss of enterocytes, we normalized MGA messages to additional messages specific for the enterocyte. The first was villin, a structural protein required for formation of microvilli on the enterocyte luminal surface (77). The second was SGLT 1, the luminal glucose transporter located on the microvillus membrane (20). The villin message and SGLT 1 correlated (r = 0.69 and r = 0.53, respectively;n = 15) with villous atrophy. Normalizing villin to β-actin mRNA improved its correlation to atrophy (r = 0.76), but normalizing SGLT 1/β-actin did not. From this analysis, it became clear that expression of the structural protein villin message level is a function of villous atrophy and that the functional protein SGLT 1 message was regulated more independently.
The functional messages for the carbohydrate hydrolases and the glucose transporter message concentrations (nanograms amplimer/nanograms RNA template) were normalized to the structural message for villin (Table 3). Values recalculated from our prior publication are indicated by (1) :
• MGA/villin message ratio was unchanged by malnutrition (P = 0.248).
• SI (1) /villin message ratio was significantly increased by malnutrition (P = 0.026).
• The mean SGLT 1/villin message ratio was conserved in malnutrition (P = 0.154).
• The previously deduced reduction in lactase mRNA (1) /enterocyte was directly confirmed by the reduced LPH/villin message in malnutrition (P = 0.043) (1).
• The increases in the SI/villin ratios led to a discovery that some messages may be enhanced in the surviving enterocytes of malnourished infants.
• The conserved or enhanced MGA, SI, and SGLT 1 message levels are in striking contrast to the 40% reduction in the enterocyte LPH/villin message level, and clearly support the hypothesis that sucrase and maltase activities are reduced in malnourished infants by loss of enterocytes. The unexpected discovery of upregulation of sucrase/villin message suggests that compensatory diet-sensitive transcriptional responses may occur in the enterocyte that regulate the proteins responsible for the hydrolysis and absorption of sugar, starch, and glucose. This is consistent with a hypothesis proposed by Pappenheimer (10), which linked the expression of brush border hydrolases to the microvillus membrane transporters.
The malnourished infants were studied while they were fed a diet of formulas fortified with sucrose and starch and complemented with cereals and fruit. There was no obvious relationship between the nature of the diets and maltase activities and message levels. All infants were experiencing increasing weight but at an unsatisfactory rate of gain. If the reduced maltase activity depicted in Table 4 is representative of the whole small intestine, it appears that the average message expression and enzyme activity of MGA, crucial for terminal starch digestion, was reduced to approximately 40% of normal. The SGLT 1 message, the final phase of starch assimilation, averaged 66% of normal. These calculations should be tested directly in this population. It has been intuitively believed, but is now demonstrated by experimental evidence, that the activities that determine essential pathways for starch α-exo-hydrolysis and glucose transport are sensitive to losses of enterocytes.
The conserved or enhanced enterocyte glucose hydrolases and transporter also suggest the possibility of a potential bottleneck at the level of enterocyte transport of the summed glucose produced by the carbohydrolases. This leads to the speculation that severe villous atrophy may lead to an excess of glucose production from a mixed diet, by combined lactase (1), sucrase (1), and maltase hydrolytic activities, and a relative inadequacy of glucose transporters. The excess glucose produced could be backflushed into the lumen and malabsorbed by the small intestine, or because of excessive glucose concentrations in the unstirred layer of the brush border, could be forced through paracellular pathways (10). This potential excess paracellular osmolar load, because of hydrolase–transporter mismatch, may contribute to ongoing basolateral ballooning and shedding of enterocytes (56–65) and to the persistence of villous atrophy. This atrophic process could prolong the reductions of maltase and sucrase, contribute to limited small intestinal starch and sugar digestion and assimilation and, because of normal inefficiency of carbohydrate assimilation and oxidation in the colon, to reduced weight gains with the usual dietary energy intakes.
There has been a recent trend to use either whole starch or malted starch in oral hydration solutions for infants with acute diarrhea, and malted starch for the refeeding of malnourished children (4,5). In the younger infant with α-amylase developmental delay and the malnourished infant with acquired α-amylase deficiency, it is assumed by the proponents that the activity of MGA will be able to compensate for missing α-amylase activities in the digestion of starch. It is necessary to caution that the feeding of malted starch only bypasses amylase deficiency. In this investigation, we have shown that MGA is reduced by an average of 40% in malnourished infants secondary to villous atrophy. It is important that reduced brush border hydrolytic capacities, varying in severity, be considered in the prescription of mixtures of dietary starch, sugar, and lactose for refeeding of malnourished infants.
The results of quantitative measurements made of maltase activity and message levels in this study are summarized in Table 4, where the observations in the M infants are expressed as percentages of of N mean values. There is a correlation between the morphologic degree of villous atrophy and the level of villin message that justifies the use of this structural message as a more quantitative measure of the number of enterocytes. Mean maltase activity and message were reduced proportional to number of enterocytes (villin message level, Figure 1). The glucose transporter message was increased in ratio to villin message in most subjects. These data advance our hypothesis that maltase (and sucrase ) messages are reduced because of villous atrophy and suggest that MGA sucrase and glucose transport messages, although reduced in aggregate, may be conserved in surviving enterocytes. The fundamental mucosal lesion, villous atrophy, requires additional investigations because of its direct effect on starch and starch oligomer digestion by the small intestine of malnourished infants.
Dr. Andy Feste of Biochroma (Spring, TX, U.S.A.) set up the liquid chromatography system for quantitation of PCR amplimers and Ms. Jestina Mason of the Child Health Research Core Laboratory at Baylor College of Medicine performed amplimer sequencing. Mr. Scott Perkinson performed the enzyme activity assays. The authors thank Ms. Conceicao Maria L. Godoy, nutritionist; Maraci Rodrigues, M.D., and Ary Lopes Cardoso, M.D., for their excellent clinical care of the infants in this study at ICR HCFMUSP; Joao Gilberto Maksoud, M.D., of the Surgical Department of ICR HCFMUSP, for provided the tissue from infants undergoing the Kasai procedure; Dr. John Waterlow, Dr. Magdalena Araya, and Dr. Laszlo G. Kömüves for reading the manuscript and providing critical reviews; Ms. Jane Schoppe for secretarial support; and Children's Nutrition Research Center editor Leslie Loddeke for editorial support.
This work was funded in part by a research grant from Bristol-Myers. It is a publication of the U.S. Department of Agriculture/Agriculture Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas and was funded in part with federal funds from the U. S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-6-001. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
Glucose transporter mismatch; Maltase-glucoamylase; Marasmic malnutrition; Starch hydrolysis; Villous atrophy