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.     

Differential effects of protein malnutrition and ascorbic acid deficiency on histidine metabolism in the brains of infant nonhuman primates

Abstract: Male infant nonhuman primates (M. nemes-trina) born in captivity were used in the study. They were divided into three groups. The first group of three animals was fed a 20% casein diet and the second group of six monkeys received a 2.0% casein diet. The third group of four monkeys received a 20% casein diet totally devoid of ascorbic acid for 3.5 weeks before the diet was supplemented with ascorbic acid (20 mg/kg diet). All the diets were given to the animals in two daily rations of 100 g/animal. The monkeys fed a 2% casein diet failed to grow, and after about 3.5 months showed variable degrees of edema, hypoalbuminemia, evidence of psychomotor disturbance, depressed plasma levels of many essential amino acids, and other features consistent with the diagnosis of protein-energy malnutrition. Examination of the brains revealed significant alterations in the levels of histidine (+ 172%) and homocarnosine (+ 146%) in comparison with the control well-fed monkeys. Associated with the increase in brain histidine was a marked elevation of brain histamine level. Protein deficiency also led to poor brain retention of ascorbic acid but not to the same degree observed in the ascorbic acid-deficient animals. The latter group of animals, after receiving their diet for about 8 months, demonstrated a modest elevation in the plasma levels of most amino acids in comparison with controls. Ascorbic acid deficiency elicited a significant reduction (p < 0.01) in brain level of histidine, with hardly any change in homocarnosine level. In addition, vitamin C deficiency produced elevation of brain histamine level comparable to findings in the protein-energy-deficient monkeys. The results suggested that protein deficiency raised brain histamine level mainly through increased availability of the precursor amino acid histidine, while defective degradation might account for the increased brain level of this amine in ascorbic acid-deficient monkeys. Histamine has been proposed to have a predominantly depressant action on relevant neurons, and has also been shown to participate with other neuro-transmitters in influencing the function of the pituitary gland by regulating release of the hypothalamic hormones into the portal vessels. The relevance of the findings of marked increases in brain histamine in experimental protein and ascorbic acid deficiencies to the behavioral and extensive endocrinological alterations seen in human malnutrition deserves some intensive investigation.     

Concentrations of histamine in brain of guinea pig and rat during dietary protein malnutration (Short Communication)

Protein malnutrition produced marked elevations in brain contents of histamine in guinea pigs (+142%) and rats (+257%) in comparison with findings in the control adequately fed animals. Associated with accumulation of this biogenic amine in each set of animals was an increase of similar magnitude in brain content of free histidine. It was concluded that the elevated brain content of histamine in protein malnutrition was due mainly to increased availability of the precursor amino acid, and that such alterations in amounts of neurotransmitter amines in the central nervous system might underlie some of the behavioural abnormalities seen in malnourished animals.     

The hypophyseal-adrenal axis in the protein-calorie malnourished rat

Male rats were fed an 8% low protein diet for 30 consecutive days beginning at 20 days of age. On day 50, the effect of the diet was evaluated on the hypophyseal-adrenal axis and the observations compared with those noted in age-matched controls fed a standard laboratory diet. The malnourished animals had significantly larger adrenal gland weights, relative to body size, and higher levels of serum corticosterone. Additionally, the number of ACTH-secreting cells in the pituitary glands of these rats was 63.8% greater than that present in the controls. These results suggest that ACTH secretion and adrenocortical physiology are stimulated under conditions of experimentally induced protein-calorie malnutrition.     

SUPPLEMENTATION OF A WHEAT BREAD DIET WITH LYSINE AND THREONINE: EFFECTS UPON INTRAUTERINE GROWTH AND BRAIN BIOCHEMISTRY

Abstract— During pregnancy, rats were fed either a standard chow diet, a wheat bread diet or a wheat bread diet supplemented with lysine and threonine. At birth, the offspring of the dams fed the supplemented diet differed from the chow fed animals only in having a smaller amount of protein in the combined cerebellum and brain stem area while the pups whose dams were fed the bread diet showed lower brain weights, deficits in protein in all brain parts and deficits in DNA in the cerebrum.     

The association of lesion-induced reductions in brain monoamines with alterations in striatal carbohydrate metabolism

Newly-weaned male guinea pigs were fed an ascorbic acid-deficient diet ad libitum and compared with control animals pair-fed an adequate diet for a similar duration. The ascorbic acid-deficient animals demonstrated prominent elevations in serum concentrations of tyrosine (+427%), phenylalanine (+36%) and arginine (+21 %) with concomitant depressions in levels of glycine (–57 %), histidine (–39 %), ethanolamine (–38%) and glutamic acid (–22 %). With few exceptions, the alterations in the liver amino acid profiles were in the same directions as those observed in the serum. The scorbutic brains showed 28–36 per cent of the retention of total ascorbic acid found in control animals and were characterized by marked elevation (+83%) in tyrosine content, hardly any alteration in phenylalanine (–9%), and depressed levels of histidine (–33 %), arginine (–25%), phosphoserine (–50%) and GABA (–12%). The implications of such abnormal changes in free amino acid patterns were evaluated in the light of the role of some of these amino acids as precursors for the synthesis of neuroregulatory substances. No difference was observed in the brain polysomal profiles as isolated from the two groups of animals. Incubation of polysomes from ascorbic acid-deficient brains with autologous pH 5 enzyme derived from cell sap not passed through Sephadex G-25 revealed low uptake of [¹⁴C]phenylalanine in comparison to that for a similar system from control animals. Use of pH 5 enzymes prepared from Sephadex-treated and dialysed cell saps eliminated the difference in specific activities of the two groups of ribosomes, an observation suggesting that ascorbic acid deficiency either intensified the activity of the inhibitory components or reduced the low molecular weight stimulatory substances present under normal conditions in the brain postmicrosomal fraction.     

Effects of diabetes and dietary manipulation on rat parotid gland secretory response to sympathetic nerve stimulation.

1. The effects of streptozotocin-diabetes, bulk-diet, low protein diet (8%) and protein-calorie malnutrition on parotid gland response to sympathetic nerve stimulation were studied in male Wistar rats. 2. Mean body weights were considerably less in diabetic and protein-calorie malnourished rats than in the other groups, but parotid gland weight was reduced only in animals placed on a low-protein diet. 3. Salivary flow rate (microliter/min/g tissue) and total protein output (mg secreted/g tissue) were reduced in diabetic rats. 4. Salivary composition was altered in diabetes and protein-calorie restriction, and the specific changes were unique to each condition. 5. Thus, with the possible exception of gland weight the effects of diabetes on parotid gland structure and function are not related to either hyperphagia or nutritional status.     

Mild prenatal protein malnutrition increases alpha2C-adrenoceptor density in the cerebral cortex during postnatal life and impairs neocortical long-term potentiation and visuo-spatial performance in rats

Mild reduction in the protein content of the mother's diet from 25 to 8% casein, calorically compensated by carbohydrates, does not alter body and brain weights of rat pups at birth, but leads to significant enhancements in the concentration and release of cortical noradrenaline during early postnatal life. Since central noradrenaline and some of its receptors are critically involved in long-term potentiation (LTP) and memory formation, this study evaluated the effect of mild prenatal protein malnutrition on the alpha2C-adrenoceptor density in the frontal and occipital cortices, induction of LTP in the same cortical regions and the visuo-spatial memory. Pups born from rats fed a 25% casein diet throughout pregnancy served as controls. At day 8 of postnatal age, prenatally malnourished rats showed a threefold increase in neocortical alpha2C-adrenoceptor density. At 60 days-of-age, alpha2C-adrenoceptor density was still elevated in the neocortex, and the animals were unable to maintain neocortical LTP and presented lower visuo-spatial memory performance. Results suggest that overexpression of neocortical alpha2C-adrenoceptors during postnatal life, subsequent to mild prenatal protein malnutrition, could functionally affect the synaptic networks subserving neocortical LTP and visuo-spatial memory formation.     

Serotonin uptake in the central nervous system of rats fed a corn-diet

1. Endogenous serotonin (5-HT), 5-hydroxyindol acetic acid (5-HIAA) content and exogenous 5-HT uptake (Km and Vmax) were measured in different brain regions (cerebellum, diencephalon, brain stem and telencephalon) of rats fed with a corn diet and restricted protein (8%) diet during 6 weeks. 2. A reduction of 5-HT levels was found in all regions studied of animals fed a corn diet, whereas, 5-HIAA was only decreased in brain stem and diencephalon. 3. An important increase in Km and Vmax were registered in brain stem and diencephalon of protein restricted animals, whereas, an increase of 5-HT uptake affinity in cerebellum, brain stem and telencephalon (35, 42 and 33% respectively) was observed. Simultaneously, under corn diet conditions, the Vmax decreased 40, 30 and 34% respectively in those regions. 4. It is suggested that the brain stem was the more sensitive area under nutritional restricted conditions and the development of some possible compensatory mechanisms of the 5-HTergic system is discussed.     

EFFECTS OF DIFFERENT LEVELS OF DIETARY PROTEIN ON BRAIN GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN YOUNG ALBINO RATS

Abstract— Studies were carried out to identify the minimum levels of protein (casein) needed in the diet in order to prevent or reverse the deficits in brain enzymes previously found with protein deficiency. Groups of weanling albino rats were fed diets containing variable amounts of protein (5, 8, 10, 15 or 20 per cent in experiment I, and 5, 6, 7, 8 or 20 per cent in experiment II) for 5 or 10 weeks. Deficits in brain wt and brain glutamate dehydrogenase and decarboxylase were found to be prevented by a diet containing 8 per cent or more of protein, although for optimum growth 15 per cent protein in the diet was found to be necessary. Groups of rats were fed a 5 or 20% protein diet for 10 weeks after which the 5% protein animals were either continued on the diet for another 10 weeks or changed to one containing 8, 10, 15 or 20% protein. The brain enzyme deficits found with the 5% protein diet were found to be fully reversed by feeding a 10% protein diet during rehabilitation.     

Effect of excess dietary histidine on rate of turnover of65Zn in brain of rat

The effect of the chronic administration of histidine on the brain zinc level was examined in growing, male Wistar rats. Using a purified diet, the minimum zinc requirement for normal growth and normal plasma and tissue zinc levels was found to be around 10 ppm. Given this zinc content; the diet was supplemented with 5% and 8% histidine, respectively, or with 10% glycine (as control). Brain zinc was analyzed by measuring the rate of turnover of⁶⁵Zn from 2–4 weeks after a single injection of the tracer. Feeding the diet supplemented with 5% histidine caused a small decrease in the plasma zinc concentration and a slight increase in the rate of turnover of⁶⁵Zn in the cerebrum and the cerebellum as compared to the control group. The animals fed the diet supplemented with 8% histidine became severely zinc deficient (as evidenced by a 50% reduction in the plasma zinc content), however, the rate of turnover of⁶⁵Zn in all brain regions examined was significantly decreased as compared to the control group. The results indicate that histidine has no specific complexing action on the brain zinc.     

ACTIVITY OF 2'',3''-CYCLIC-NUCLEOTIDE 3''-PHOSPHODIESTERASE IN REGIONS OF RAT BRAIN DURING DEVELOPMENT: QUANTITATIVE RELATIONSHIP TO MYELIN BASIC PROTEIN

2′,3′-Cyclic-nucleotide-3′-phosphodiesterase activity was examined in several regions of rat brain during development, namely optic nerve, olfactory bulb, cerebrum, cerebellum, midbrain, brain stem, and spinal cord. From 4 to 120 days the total activity increased in all regions, although the specific activity approached a constant value in adults. The developmental profile of the enzyme appeared to correlate with the onset of myelination and with the levels of myelin basic protein as well as the appearance of galactocerebroside sulfotransferase. A correlation coefficient of 0.91 was found between total basic protein, expressed as the per cent of the adult (120 day) value, and total enzyme activity over 12–42 days of age (P < 0.001) from six different brain regions as well as for whole brain. By increasing the sensitivity of the assay with the use of [³H-8]adenosine 2′,3′-cyclic monophosphate, we were able to detect activity at birth in both whole brain and spinal cord.     

Myelin Lipids in the Developing Cerebrum, Cerebellum, and Brain Stem of Normal and Undernourished Children

Abstract: The developmental lipid profiles in the human cerebrum, cerebellum and brain stem are presented, with special reference to galactolipids as myelin markers to trace myelination in the three main parts of the human CNS. A group of undernourished children were also studied to test the vulnerability of myelinogenesis in the different regions of the human brain. Myelination was well advanced in the brain stem with regard to the other brain regions, a fact reflected in the much higher concentration of myelin lipids in the brain stem of the human foetus of 26 weeks of gestational age. The cerebrum, on the other hand, had the lowest galactolipid concentration during the prenatal period, galactolipid levels in the cerebellum being four times higher. From just before the end of gestation the accretion of galactolipids accelerated enormously in the cerebrum, whereas it slowed down considerably in the cerebellum. Consequently, in relation to prenatal levels galactolipids increased most rapidly in the cerebrum, followed by the cerebellum and finally by the brain stem. These regional differences were in clear contrast to data from the rat, as was the finding that only the cerebrum of undernourished children had a galactolipid concentration significantly decreased with respect to normal values. A relationship between the different myelination patterns in the human and the rat and the distinct vulnerability of myelinogenesis in the two species is suggested.     

THE EFFECT OF A LOW PROTEIN DIET AND EXOGENOUS INSULIN ON BRAIN TRYPTOPHAN AND ITS METABOLITES IN THE WEANLING RAT

—Male Wistar rats aged 24 days were divided into three groups. Two groups were given a high protein (250 g/kg casein) and a low protein (30 g/kg casein) diet respectively. The third group was given an amount of the high protein diet containing the same amount of energy as that consumed by the low protein diet rats. The plasma of the animals on low protein contained 20% of the concentration of tryptophan of animals on the other two diets. In these animals the concentration of tryptophan was reduced in the forebrain, cerebellum and brain stem, and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were reduced in the forebrain and brain stem. The low protein diet decreased the total uptake of l -[G-³H]tryptophan into the brain and its incorporation into brain protein. Plasma insulin concentrations were reduced in the low protein and ‘restricted high protein’ animals and the plasma corticosterone concentration was raised in the low protein animals. Exogenous insulin did not raise the plasma tryptophan concentration in the low protein animals but it increased the uptake of l -[G-³H]tryptophan into the brain and its incorporation into protein. Rehabilitation for 7 days restored the plasma and brain tryptophan concentrations and those of brain 5-HT and 5-hydroxyindoleacetic acid to control values.     

Experimental protein malnutrition in primates--cytochemistry of the nervous system

Experimental protein malnutrition was induced in groups of young juvenile squirrel monkeys by feeding them ad libitum diets very low in protein content, whereas a diet containing 25% protein content was fed to the control animals. Detailed cytochemical studies have clearly shown the sensitivity of the nervous system to dietary abuse. The motor neurons of the spinal cord and the Purkinje cells of the cerebellum are very sensitive to protein deficiency, and the number of oligodendroglia cells increases sharply. Gallocyanin stained preparations from the malnourished animals show significant decrease in the amount of RNA in the Purkinje cells of cerebellum and the anterior horn cells of the spinal cord. The larger neurons show prominent chromatolysis with concomitant increase in the number of oligodendrocytes surrounding such nerve cells. The different layers of the cerebellar cortex of the malnourished animals, as well as some of the neurons of the spinal cord, showed decreased activity of succinate dehydrogenase and increased levels of thiamine pyrophosphatase and lactate dehydrogenase. Although ATPase activity appeared to remain unchanged quantitatively, it showed profound disturbance in its in situ localization, especially in the mitochondrial ATPase located in the cytoplasm. The enzyme activity in the nuclear membrane appeared unchanged. These studies emphasize the importance of studying in situ changes in the anatomically heterogeneous nervous tissue, rather than of working only with biochemical methods using homogenized material.     

Effects of cysteine on amino acid concentrations and transsulfuration enzyme activities in rat liver with protein-calorie malnutrition

The changes in amino acid concentrations and transsulfuration enzyme activities in liver were investigated after 4-week fed on 23% casein diet (control group) and 5% casein diet without (protein-calorie malnutrition, PCM group) or with (PCMC group) oral administration of cysteine, 250 mg/kg (twice daily, starting from the fourth week) using rats as an animal model. By supplementation with cysteine in PCM rats (PCMC group), cysteine level was elevated almost close to the control level, and glutathione (GSH), aspartic acid and serine levels were restored greater than the control levels. The measurement of transsulfuration enzyme activities exhibited that gamma-glutamylcysteine ligase (gamma-GCL) activity was up-regulated in rats with protein restriction (PCM group), and cysteine supplementation (PCMC group) down-regulated to the control level. One-week supplementation of cysteine (PCMC group) significantly down-regulated the cysteine sulfinate decarboxylase activity. These results indicate that the availability of sulfur amino acid(s) especially cysteine appears to play a role in determining the flux of cysteine between cysteine catabolism and GSH synthesis.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

Protein-energy malnutrition and micronutrient deficiencies may down-regulate immune response and increase morbidity and mortality due to infection. In this study, a murine model was used to study the effects of protein, iron and zinc deficiencies on the immune response to Leishmania (Leishmania) chagasi infection. Mice were initially fed a standard diet or with a diet containing 3% casein but deficient in zinc and iron. After malnutrition was established, mice were inoculated with L. chagasi and sacrificed four weeks later in order to evaluate liver and spleen parasite loads and serum biochemical parameters. Significant decreases in liver and spleen weight, an increase in the parasite loads in these organs and decreases in serum protein and glucose concentrations in malnourished animals were observed. Furthermore, the production of interferon-gamma by spleen cells from infected malnourished mice stimulated by Leishmania antigen was significantly lower compared with that in control diet mice. These data suggest that malnutrition alters the immune response to L. chagasi infection in the BALB/c model and, in association with the effects on biochemical and anatomical parameters of the host, favored increases in the parasite loads in the spleens and livers of these animals.