Bonnie S. Worthington

Effect of daily ethanol ingestion on intestinal permeability to macromolecules

The effects of regular ethyl alcohol ingestion on morphological and permeability characteristics of the small intestine were assessed in mature rats using the tracer protein, horseradish peroxidase. Thirty adult rats were divided into two groups and provided a standard commercial diet in pellet form. Each morning, after an overnight fast, every animal in the experimental group was administered by gavage an aliquot of 20% ethanol; animals in the control group were provided aliquots of 20% sucrose in water by the same method. After 4 and 8 weeks on the gavage routine (and 10 days and 4 weeks after gavage cessation), jejunal permeability to horseradish peroxidase was examined in animals from each group. Using a routine ligated-loop procedure and light and electron microscopy, ethanol-exposed rats demonstrated increased intestinal permeability to horseradish peroxidase by 4 weeks; sucrose-exposed animals revealed little alteration in mucosal integrity. It is proposed that regular ingestion of sizable amounts of alcohol alters morphological characteristics of the gut and increase the permeability of the mucosa to “undigested” macromolecules.

Accumulation of histidine, 3-methylhistidine, and homocarnosine in the brains of protein-calorie deficient monkeys.

Abstract— Seventeen monkeys (M. nemestrina and M. fascicularis) aged 10 months to about 5 yr were divided into two groups and fed either an adequate protein diet (20% casein) or a low‐protein diet (2% casein). The diets were supplied to the animals in restricted amount (200 g/animal in two daily rations). In one experiment, the malnourished animals were initially fed a diet containiing 8 per cent protein and the protein content of the diet was gradually reduced over a period of 9 months, to 2 per cent. After about 3 months on the 2 per cent protein diet, the malnourished monkeys showed growth failure, severe anorexia, peri‐ocular oedema, tremors of the head and limbs, atrophy of several visceral organs, fatty liver, hypoalbuminaemia, and depressed serum levels of many essential amino acids with an elevation of the ratio of non‐essential to essential amino acids. These features are consistent with protein‐calorie malnutrition. Examination of the brains revealed significant alterations in the levels of glycerophosphoethanolamine (—40 per cent), glutamic acid (—25 per cent), histidine (+230 per cent), homocamosine (+185 per cent), 3‐methyl‐histidine (+147 per cent), lysine (+55 per cent), phenylalanine (+33 per cent) and tyrosine (+26 per cent) in comparison to findings on the well‐fed monkeys. The possible implications of elevated cerebral contents of homocarnosine in malnourished monkeys are discussed in the light of several reported human cases in whom neurological disorders are associated with increased histidine‐containing dipeptides in the brain, CSF, blood and urine.

Elevation of brain histamine content in protein-deficient rats.

—Male rats of the Sprague‐Dawley strain (80–250 g body wt) were fed either an adequate protein diet (18% lactalbumin) or a protein‐deficient diet (0.5% lactalbumin). After 5–8 weeks of receiving the low protein diet, some of the malnourished rats were rehabilitated with an adequate protein diet. The malnourished rats exhibited significant elevations in brain levels of histidine (+415%) and homocarnosine (+100%) in comparison to findings in the control animals of similar age. Associated with the elevated brain levels of histidine in malnutrition was a prominent increase in brain content of histamine (+ 150‐+ 238%). The mean brain histamine levels (ng/g) in the control rats varied from 45.96 to 56.15 in several experiments. In the protein‐deficient rats, values ranged from 115 to 190. Refeeding the malnourished rats with adequate protein diet elicited reversal of histidine and histamine levels to near normal values within 1 week. The increased brain content of histamine in malnutrition was attributed to enhanced rate of production resulting from increased availability of the precursor amino acid, a conclusion consistent with elevation also of the brain content of homocarnosine (γ‐aminobutyryl‐l‐histidine) which is another major route of disposal of histidine in the brain. The relevance of these neurochemical alterations to the behavioural changes often associated with protein malnutrition, deserves some intensive examination.

Regional distribution of homocarnosine and other ninhydrin-positive substances in brains of malnourished monkeys.

—Eight male monkeys (Macaca nemestrina) aged 6–9 months were divided into two groups and fed either an adequate protein diet (20% casein) or a protein deficient diet (2% casein). After 3‐ 5 months of receiving the low protein diet, the malnourished monkeys showed extensive fatty metamorphosis of the liver cells, distorted patterns of plasma and hepatic free amino acid pools, and other features consistent with the diagnosis of protein‐calorie malnutrition. Examination of the cerebrum, cerebellum and brain stem in the malnourished animals revealed profound accumulation of 3‐methylhistidine, histidine and homocarnosine in all three regions. For histidine, the cerebral, cerebellar and brain stem levels in the protein deficient animals increased by 145, 104 and 101 per cent over levels observed in corresponding regions of the brain in well‐fed monkeys. Similarly, there were significant elevations in homocarnosine contents of the cerebrum (+ 99 per cent), cerebellum (+ 140 per cent) and brain stem (+ 146 per cent) in comparison to levels in control animals. In contrast, the levels of valine, serine and aspartic acid were markedly reduced in all three brain areas in the malnourished animals. Protein‐calorie deficiency also produced reductions in the brain levels of taurine, glutamic acid, isoleucine, leucine and threonine which varied in magnitude in the three major regions of the brain examined. These biochemical alterations which may in part underlie some of the psychomotor changes often observed in protein‐calorie malnutrition, were discussed not only in relation to the role of amino acids as precursors for the synthesis of neuroregulatory substances but also with due regard to the possibility that some of these ninhydrin‐positive substances such as GABA, homocarnosine, glycine and the dicarboxylic amino acids may possess neuroexcitatory or inhibitory properties in various parts of the central nervous system.

The influence of protein malnutrition on ileal permeability to macromolecules in the rat

Ileal absorption of intact protein was assessed in groups of control and protein-deficient rats during a 5-month period of dietary protein restriction. At monthly intervals following diet initiation, 3 animals from both the control and deficient groups were administered horseradish peroxidase via ligated ileal loops either 15, 45 or 90 minutes before loop excision and processing for light and electron microscopy. Morphological evaluations revealed no change in pinocytotic activity during the 5 months of progressive protein malnutrition. After 3 months, however, partial deterioration of mucosal structure was observed with apparent separation of apical intercellular junctions and movement of peroxidase molecules directly between cells. These observations in the ileum are compared to previous findings in the jejunum. It is suggested that ileal and jejunal absorptive cells respond uniquely to protein deprivation but that eventual junctional deterioration occurs in both regions with potential detrimental consequences for the host.

Absorption of intact protein by colonic epithelial cells of the rat

Colonic absorption of intact protein was examined in adult rats using histological and ultrastructural procedures. Horseradish peroxidase was introduced into ligated colonic loops and retained therein for 5, 10, 20, or 30 minutes prior to excision of the loops, and their processing for microscopy. Morphological evaluation revealed evidence of peroxidase absorption via pinocytosis. Tracer particles were observed adherent to the mucosal border, in apical pinocytotic vesicles, in vesicles adjacent to and fusing with lateral and basal cell membranes, in extracellular spaces throughout the mucosa, in lymphatic channels of the submucosa, and occasionally in blood capillaries of mucosal and submucosal regions. The significance of these findings is discussed in light of the frequent presence of dietary and/or microbial macromolecules in the luminal milieu of the large intestine. It is suggested that pinocytotic uptake and subsequent vesicular transport and exocytosis of intact protein may occur in the colon of some species, and that such a phenomenon may be responsible for penetration of the mucosal barrier by macromolecular antigens or toxins.