Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments.

Cyanidin 3-glucoside improves diet-induced metabolic syndrome in rats

Increased consumption of dark-coloured fruits and vegetables may mitigate metabolic syndrome. This study has determined the changes in metabolic parameters, and in cardiovascular and liver structure and function, following chronic administration of either cyanidin 3-glucoside (CG) or Queen Garnet plum juice (QG) containing cyanidin glycosides to rats fed either a corn starch (C) or a high-carbohydrate, high-fat (H) diet. Eight to nine-week-old male Wistar rats were randomly divided into six groups for 16-week feeding with C, C with CG or QG, H or H with CG or QG. C or H were supplemented with CG or QG at a dose of ∼8 mg/kg/day cyanidin glycosides from week 8 to 16. H rats developed signs of metabolic syndrome including visceral adiposity, impaired glucose tolerance, hypertension, cardiovascular remodelling, increased collagen depots in left ventricle, non-alcoholic fatty liver disease, increased plasma liver enzymes and increased inflammatory cell infiltration in the heart and liver. Both CG and QG reversed these cardiovascular, liver and metabolic signs. However, no intact anthocyanins or common methylated/conjugated metabolites could be detected in the plasma samples and plasma hippuric acid concentrations were unchanged. Our results suggest CG is the most likely mediator of the responses to QG but that further investigation of the pharmacokinetics of oral CG in rats is required.

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

Effect of the antitumor antibiotic chromomycin A3 on the humoral immune response in rats

Chromomycin A3 (250 mug/kg) suppressed the humoral immune response in rats against sheep erythrocytes when administered 48 h or later after antigenic stimulus. The antibiotic at this dose enhanced immunity when given along with or before antigen administration. The natural heterohemagglutinin levels in rabbits and guinea pigs were not affected by the antibiotic (10 mug/kg per day x 7).

Antitumor immunity in rats against Yoshida ascites sarcoma after treatment with antibiotics

Chromomycin A3, mithramycin, olivomycin and actinomycin D, four antitumor antibiotics inhibit growth of Yoshida ascites sarcoma (YAS). The antibiotic treated tumor-free rats exhibit antitumor immunity as judged by rejection of subsequent tumor transplant. The lymphocytes from immune rats are able to inhibit tumor growth in syngenic animals.

The onset of oestrus and ovulation in lactating rats

Lactation delays the re-initiation of oestrous cyclicity in rats, resulting in physiological sterility for the duration of suckling. During this phase, the secretion of pituitary gonadotrophins is suppressed by an unknown mechanism. Continued application of the suckling stimulus by litter replacement (Bruce, 1958; Nicoll & Meites, 1959), or injections of prolactin (Meites & Nicoll, 1959), have been shown to prolong lactation considerably beyond the usual period. The present study aimed to demonstrate the role of prolactin in inhibiting the gonadotrophin secretion necessary for the re-establishment of oestrous cyclicity during lactation. Pregnant rats weighing approximately 300 g were obtained from the Institute colony and housed in individual cages. At parturition, the number of young in the litter was adjusted to eight, two or one as required. The day following the post-partum oestrus was regarded.

The role of fsh and lh in the initiation of ovulation in rats and hamsters: a study using rabbit antisera to ovine fsh and lh

The relative rôles of FSH and LH in ovulation induction in immature and adult cycling rats and hamsters have been evaluated. Both heterologous purified pituitary hormones and homologous crude pituitary extracts have been used as ovulatory stimuli in immature animals primed with PMSG. Well-characterized FSH and LH antisera have been used in the above model systems to achieve specific neutralization of FSH and LH. The present study revealed that LH is the physiological trigger needed for induction of ovulation in both rats and hamsters and FSH cannot, by itself, induce ovulation in the total absence of LH.

Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens.

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8'-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8'-apocarotenol, 8'-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.

Studies on the metabolism of β-carotene and apo-β-carotenoids in rats and chickens

1. (1) The relative abilities of the various cell fractions of rat and chicken liver to oxidize and reduce retinal and 8'- and 12'-apo-β-carotenal were investigated and it has been shown that, while retinal is exclusively oxidized by the soluble fraction, the apocarotenals are mostly oxidized by the particulate fractions of the homogenate. 2. (2) Addition of NAD+ or NADP+ markedly activated the oxidation of the apocarotenals, but not of retinal by the particulate fractions. 3. (3) Considerable amounts of retinal and 8'-, 10'- and 12'-apo-β-carotenal were isolated from the intestine of chickens fed β-carotene and these apocarotenoids were conclusively identified. 4. (4) Significant amounts of 8'-, 10'- and 12'-apo-β-carotenoic acids were isolated from the intestine of rats given 8'-apo-β-carotenal and these apocarotenoic acids were also conclusively identified. 5. (5) In the light of these observations it is suggested that during conversion to vitamin A, the β-carotene molecule is simultaneously attacked by the dioxygenase at several double bonds, the primary attack being at the central double bond and a tentative scheme for the mechanism of conversion is proposed.

Metabolic fate of menthofuran in rats. Novel oxidative pathways

Metabolic fate of menthofuran (II) in rats was investigated. Menthofuran (II) was administered orally (200 mg/kg of the body weight/day) to rats for 3 days. The following metabolites were isolated from the urine of these animals: p-cresol (VI), 5-methyl-2-cyclohexen-1- one (VII), 3-methylcyclohexanone (VIII), 3-methylcyclohexanol (IX), 4- hydroxy-4-methyl-2-cyclohexen-1-one (V), geranic acid (XI), neronic acid (XII), benzoic acid (XIII), and 2-[2'-keto-4'- methylcyclohexyl]propionic acid (X). Incubation of menthofuran (II) with phenobarbital-induced rat liver microsomes in the presence of NADPH and oxygen resulted in the formation of a metabolite tentatively identified as 2-Z-(2'-keto-4'-methylcyclohexylidene)propanal (III; alpha,beta-unsaturated-gamma-keto-aldehyde). The structure assigned was further supported by trapping this metabolite (III) as a cinnoline derivative. Phenobarbital-induced rat liver microsomes also converted 4- methyl-2-cyclohexenone (IV) to 4-hydroxy-4-methyl-2-cyclohexenone (V) and p-cresol (VI) in the presence of NADPH and oxygen. On the basis of both in vivo and in vitro studies, a possible mechanism for the formation of p-cresol from menthofuran has been proposed.