Effects of peroxidative stress and age on the concentration of a deoxyguanosine-malondialdehyde adduct in rat DNA.

The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had no effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-alpha-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain >> liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.

Calcium glycerophosphate as a source of calcium and phosphorus in total parenteral nutrition solutions.

Calcium glycerophosphate (CaGP) was tested as an alternative to calcium gluconate (CaGluc) and potassium mono- and dibasic phosphate (KPhos) as a source of Ca and P in total parenteral nutrition (TPN) solutions for piglets. Four-day-old piglets were infused for 7 days with a TPN solution that provided either 4.2 mmol Ca and 2.1 mmol P/kg/24 h as CaGluc and KPhos (the maximum quantities that can be provided using these sources), or 15.0 mmol Ca and 15.0 mmol P/kg/24 h as CaGP. Ca and P retentions were more than six times greater (p less than 0.01) in the piglets receiving CaGP (14.5 +/- 0.2 vs 2.2 +/- 0.3 mmol Ca/kg/24 h and 13.3 +/- 0.4 vs 2.4 +/- 0.1 mmol P/kg/24 h) (Mean +/- SEM). The ratio of Ca to fat-free dry weight, an indicator of bone mineralization, was significantly higher (p less than 0.05) in the humerus (174.8 +/- 2.2 vs 147.2 +/- 6.7) and femur (158.3 +/- 4.8 vs 130.1 +/- 7.8) in the CaGP group. This study showed that CaGP is efficiently used as a source of Ca and P in TPN solutions for piglets. The results suggest that the use of CaGP as the source of Ca and P in TPN solutions may prevent the development of the undermineralized bone seen in low-birth weight infants nourished intravenously.

Sources of protein-induced endogenous acid production and excretion by human adults.

The origin of the increase in endogenous acid production and excretion associated with the calciuretic action of high protein intakes was investigated in human adults. Eight subjects, 4 males and 4 females, aged 25-36 years, were fed a low protein diet (50 g/day) and a high protein diet (120 g/day in males and 106 g/day in females) for 7 days each. The high protein diet was formulated by supplementing the low protein diet with a mixture of four purified proteins. Increased protein intake was associated with increases in urinary Ca, sulfate, titratable acidity (acid phosphates) and ammonium, and decreases in urinary pH and bicarbonate. There was no increase in organic anion excretion. The increases in endogenous acid production (EAP) and net acid excretion (NAE) were entirely attributable to the oxidation of excess sulfur amino acids (SAA), which yields 2 moles of hydrogen ions per mole of amino acid catabolized. The results differ in this respect from those reported for studies on the effect of SAA loading, which indicate that non-SAA make a major contribution to the increase in EAP seen under these conditions.

Malondialdehyde excretion by subjects consuming cod liver oil vs a concentrate of n-3 fatty acids.

Urinary malondialdehyde (MDA), an indicator of lipid peroxidation in the diet and in the tissues, was determined in human adults consuming a supplement of n-3 fatty acids derived from a pharmaceutical grade of cod liver oil (CLO) without added antioxidants vs a concentrate of n-3 acids containing dodecyl gallate and vitamin E. MDA excretion increased immediately in the subjects consuming CLO but remained unchanged in those ingesting the concentrate for 50 days. The increase in the subjects taking CLO was attributable to MDA in the oil. The results indicate that consuming unstabilized fish oils as a source of n-3 fatty acids may entail exposure to potentially toxic products of lipid peroxidation.

Vitamin D metabolism in the hooded seal (Cystophora cristata).

Fish-eating mammals, such as seals, appear to ingest levels of vitamin D that are toxic to most mammals. To determine how seals cope with high vitamin D intakes, the metabolism of tritiated cholecalciferol ([3H]D3) was investigated in hooded seal (Cystophora cristata) pups during their postweaning fast and pups and adults consuming herring alone or supplemented with 400,000 iu D3 daily. [3H]D3 was metabolized to 25-[3H]OHD3 and 24,25-[3H](OH)2D3. 1,25-[3H](OH)2D3 was not detected, but plasma levels of 1,25-(OH)2D were similar to those in other mammals and were not affected by vitamin D intake. Plasma vitamin D, 25-OHD and 24,25-(OH)2D increased with vitamin D intake, but 25-OHD did not increase to the extent seen in other mammals. The supplemented seals showed no evidence of toxicity. Levels of 24,25-(OH)2D were higher in the unsupplemented seals (4 to 33 ng/mL) than reported in other mammals with similar 25-OHD levels and did not decrease with 25-OHD. High levels of 24,25-(OH)2D relative to 25-OHD have also been found in hooded seals in the wild. The half-lives of vitamin D, 25-OHD and 24,25-(OH)2D were shorter than those reported for most other mammals. Increased conversion of 25-OHD to 24,25-(OH)2D and a high capacity for vitamin D storage in their large blubber mass appeared to be factors in the resistance of seals to vitamin D toxicity.

Response of urinary malondialdehyde to factors that stimulate lipid peroxidation in vivo.

Malondialdehyde (MDA) derivatives occur as normal constituents of rat and human urine. In a previous study, it was found that MDA excretion in rats is responsive to MDA intake and to certain factors that increase lipid peroxidation in vivo: vitamin E deficiency, iron administration and a high concentration of cod liver oil (CLO) fatty acids in the tissues. In the present study, the effect on MDA excretion of several additional dietary and endogenous factors was evaluated. The composition of dietary fatty acids had a major influence on MDA excretion in fed animals, being highest for animals fed n-3 fatty acids (20:5 and 22:6) from CLO, intermediate for those fed n-6 (18:2) acids from corn oil (CO) and lowest for those fed saturated acids from hydrogenated coconut oil (HCO). Diet was the main source of urinary MDA in all groups. Fasting produced a marked increase in urinary MDA, which tended to be higher in rats previously fed CLO. Fasting MDA excretion was not affected by the level of CO in the diet (5, 10 or 15%), indicating that feeding n-6 acids does not increase lipid peroxidation in vivo. Adrenocorticotropic hormone and epinephrine administration increased urinary MDA, further indicating that lipolysis either releases fatty acid peroxides from the tissues or increases the susceptibility of mobilized fatty acids to peroxidation. A decrease in fasting MDA excretion was observed in rats previously fed a high level of antioxidants (vitamin E + BHT + vitamin C) vs a normal level of vitamin E. MDA excretion increased following adriamycin and CCl4 administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Micronutrients and cancer prevention: are the RDAs adequate?

The involvement of certain micronutrients (vitamin C, vitamin E, beta-carotene, selenium) in the antioxidant defense system against free radical cell damage, and of vitamin A in the differentiation of epithelial cells, has raised the question whether intakes of these nutrients in excess of their recommended daily allowances should be recommended to the general public for cancer prevention. The considerations surrounding this question are discussed, and it is concluded that such measures are unjustified by present epidemiological and experimental evidence. Any such action should await the outcome of ongoing intervention trials.

Self-regulation of phosphate intake in the rat: the influence of age, vitamin D and parathyroid hormone.

Growing rats offered a choice of four pairs of diets, one low in P (0.1%) and the others containing 0.3, 0.6, 1.2 or 1.8% P, selected food mixtures in each case with nearly identical P contents (0.23-0.24%) (Ca:P = 2.2:1). Mature rats offered the same dietary choices exhibited less rigid diet selection but clearly preferred a diet higher in P (0.64-0.69%) (Ca:P = 0.9:1). Vitamin D-deficient animals selected less P than controls and parathyroidectomized rats severely limited their P intake. The increase in self-determined P consumption relative to Ca in mature rats is consonant with the greater decrease in the requirement for Ca associated with maturation and cessation of bone growth. Susceptibility to hypocalcemia in vitamin D deficiency and parathyroidectomy is a probable factor in the increased sensitivity to excess dietary P, which further depresses plasma Ca. These experiments confirm the existence of a feedback mechanism that regulates the voluntary consumption of P in accordance with physiological needs.

Long-term effects of excess protein and phosphorus on bone homeostasis in adult mice.

In adult human subjects, an interaction between dietary protein and phosphorus has been reported, in which the hypercalciuric effect of excess protein is counteracted by the hypocalciuric effect of phosphorus, with restoration of calcium balance. In adult rodents, bone homeostasis is maintained over a wide range of protein intakes, whereas high phosphorus diets cause bone loss, despite their hypocalciuric effect, as the result of an overriding increase in the excretion of endogenous fecal calcium. The present study was designed to determine whether there is an interaction between dietary protein and phosphorus with respect to bone homeostasis in adult mice. Four-month-old 45Ca-labeled B6D2F1 female mice were fed for 52 weeks the following diets (in percent): control, Ca, 0.6; P, 0.3; protein, 15; high P, Ca. 0.6; P, 1.2; protein, 15; high protein, Ca, 0.6; P, 0.3; protein, 30; and high P + high protein, Ca, 0.6; P, 1.2; protein, 30. Urinary calcium was persistently increased in the high protein group, depressed in the high P group and transiently depressed in the high P + high protein group. Excess dietary protein prevented phosphorus-induced kidney calcinosis. 45Ca loss was increased in the high P groups, but not in the high protein group. There were significant decreases in the mass of the femurs and tibias in both high P groups, whereas there was no effect of high protein intake. These results show that bone homeostasis in adult mice is sensitive to excess dietary phosphorus but not to excess protein, and that there is no interaction between these nutrients with respect to their effects on bone.

Effect of a chronic acid load as sulfate or sulfur amino acids on bone metabolism in adult rats.

Diets containing an acid load as either sulfur amino acids (SAA) or inorganic sulfate were fed to 45Ca-labeled adult male rats for 10 months. Radioisotope excretion and bone composition data (femur, tibia, mandibles) were compared with those for rats fed a control (15% soy protein) diet. Rats fed the SAA supplement (1.28% cystine plus 0.19% methionine) exhibited a significant reduction in femoral weight and A:R ratio and a tendency toward lower specific gravity, dry weight, fat-free weight and calcium content. Femoral radioautographs indicated a reduction in metaphyseal bone density in six of eight animals. We have postulated that the osteopenia produced by feeding excess free SAA may be due to decreased bone formation caused by a reaction between homocysteine and the aldehyde groups of collagen, as in genetic homo-cystinuric osteoporosis. Sulfate feeding (1.42% of the diet) produced a significant increase in 45Ca excretion, indicative of enhanced bone resorption, lasting about 2 months. There was a tendency for bone mass measurements to be lower than controls after 10 months, but the differences were not significant. Two of eight sulfate-fed rats showed radiographic evidence of osteopenia. No evidence of osteopenia was seen in the controls or in rats previously fed a high protein diet containing the same concentration of SAA.

Self-regulation of phosphate intake by growing rats.

Groups of growing rats (100-150 g) were offered a choice of two diets, one containing a deficient concentration of phosphorus (0.1%) and the other containing 0.3, 0.6, 1.2 or 1.8% phosphorus. All diets contained 0.6% Ca and were isocaloric. Except for the groups that were offered the 0.1 and 0.3% phosphorus diets, all the animals selected mixtures of diets containing nearly identical phosphorus contents (0.33-0.36%). The group offered the two diets lowest in phosphorus selected 83% of their food from the higher (0.3%) phosphorus diet to obtain a mixture containing 0.25% phosphorus. Irrespective of the phosphorus content of the diets available, all groups ate similar amounts of food, made similar weight gains and maintained normal plasma levels of calcium and phosphorus. The experiment demonstrates the existence of a feedback mechanism by which growing rats regulate their phosphorus intake within narrow limits when allowed to self-select among diets of markedly different phosphorus contents. It is postulated that feedback regulation of phosphorus intake is mediated by changes in plasma calcium homeostasis.

Calcium, phosphorus, and osteoporosis.

Gross epidemiological data indicate there are no significant differences in rates of aging osteopenia among countries with substantially different amounts of Ca in their national food supplies. This-observation, plus the fact that Ca administration fails to reverse osteoporotic bone loss, has led some investigators to conclude that Ca nutrition is an insignificant factor in the etiology of osteoporosis. However, it has become apparent that a Ca intake that may be adequate for adults consuming a low protein, low P, neural, or alkaline cereal-based diet is not necessarily adequate for subjects consuming a high protein, high P, acidic mixed Western diet. Ca administration inhibits postmenopausal osteopenia and there is epidemiological evidence that a liberal Ca intake reduces bone loss in middle adulthood. Ca intakes in the United States and Canada appear generally satisfactory among children and young adults, but low intakes by many individuals of middle age is a cause for concern, especially among women. Although the Ca:P ratio for the average diet consumed in these countries (about 1:1.6) appears to be satisfactory, a low intake of dairy foods, coupled with a high intake of other foods rich in natural and added phosphorus, may raise the ratio above 1:2, a value beyond which animal studies indicate that there is a risk of increased bone loss.

A hormonal assessment of bovine parturient paresis: evidence for a role of oestrogen.

A comparative assessment was made of the hormonal control of calcium homeostasis in eight dairy cows which developed parturient paresis and in seven normal animals from the same herd. Plasma levels of calcium, phosphorus, magnesium, free hydroxyproline, 25-hydroxycholecalciferol (25-OHD), 1,25-dihydroxycholecalciferol (1,25-(OH)2D), parathyroid hormone, calcitonin, prolactin and oestrogen were monitored from 30 days prepartum to 15 days post partum. Prepartum levels of plasma calcium, hydroxyproline and calcitonin were depressed in the paretic animals, and plasma levels of phosphorus and oestrogen were elevated. Plasma levels of 25-OHD remained stable in both groups, whereas levels of 1,25-(OH)2D, parathyroid hormone and prolactin rose sharply at parturition. Plasma hydroxyproline, an index of bone resorption, began to rise 2 days prepartum in the control cows but not until 2 days post partum in the paretic cows. The data indicate that bone resorption was inhibited in the paretic group at the onset of lactation, and that a decreased capacity for bone resorption is a major factor in the susceptibility of some cows to this disease. The failure of the paretic animals to resorb bone was not associated with an inability to synthesize the calcium-mobilizing hormones parathyroid hormone or 1,25-(OH)2D, or to regulate the production of calcitonin. However, hypocalcaemia in the affected animals was associated with a significantly higher plasma level of oestrogen (a known inhibitor of bone resorption) in the immediate prepartum period. Following parturition, plasma levels of oestrogen fell rapidly and active bone resorption ensued in the paretic animals.

Effect of chronic high protein feeding on bone composition in the adult rat.

A study of the effect of feeding a high protein diet on bone metabolism was conducted using adult rats deep-labeled with 45Ca. A control diet (15% soy protein plus 0.2% methionine) and a high protein diet (control plus 20% lactalbumin) were fed for 10 months. Rats fed the high protein diet exhibited increases in urinary Ca, 45Ca, sulfate and volume. Total 45Ca excretion, urine calcium specific activity and urine phosphorus initially were depressed indicating an increase in the intestinal absorption of calcium, then were not significantly different from control values. After 10 months, analysis of the femur, tibia and mandible revealed no differences in wet weight, dry fat-free or ash weight, calcium, phosphorus, magnesium, or residual 45Ca content. Specific gravity and ash density also were unaffected by protein intake, as were femur length and midshaft cortical thickness. No changes in bone composition were found which would indicate that high protein diets promote bone loss in this species. The adult rat appears to be capable of compensating for the increased urinary loss of Ca associated with an increment in acid load (whether derived from an increase in diet acidity or in metabolic acid production) by reducing the fractional loss of endogenous Ca in the feces.

Increased 1,25-dihydroxyvitamin D3 synthesis in rats fed a high-phosphorus diet.

Rats fed vitamin D-deficient diets containing 0.6% Ca and 0.3%, 0.6%, 1.2%, or 1.8% P exhibited progressive increments of hypocalcemia and hyperphosphatemia. In vitro assay of 25-hydroxyvitamin D3-1-alpha-hydroxylase (1-alpha-hydroxylase) activity in isolated kidney cortical mitochondria showed that hyperphosphatemia in the presence of hypocalcemia was associated with an increase in enzyme activity. The results indicate that the stimulation of 1-alpha-hydroxylase associated with depressed plasma Ca in rats fed a high-P diet overrides any inhibition of the enzyme that may be caused by excess plasma phosphate.