Ribonuclease activity and isoenzymes in raw and processed cows' milk and infant formulas.

Because of evidence of an immunologic role for ribonuclease II (E.C. 3.1.27.5) in mammals, its presence in milk was further characterized to provide a basis for study of possible contributions of its activity to the health of infants. Isoenzymes of ribonuclease II were quantitatively resolved from milk samples as small as 1 ml or less by chromatography on phosphocellulose. Three isoenzymes detected in bovine milk were the previously reported ribonucleases A and B and a form termed ribonuclease II-1. These isoenzymes were in the ratio of 70:30:1. Form II-1 was unique in its inability to hydrolyze polycytidylate. Bovine colostrum contained 10 to 15 times more ribonuclease II-1 than does milk and three times more total ribonuclease II per unit volume. Human milk contains about 1% the concentration of ribonuclease II found in cows' milk. Ribonuclease II activity in milk was quite stable in the acidic conditions of whey production and during low heat treatments. However, most of its enzymatic activity was lost with high heat treatments. No commercially manufactured milk-based or soybean-based infant formula assayed contained nearly as much ribonuclease activity as either human or bovine milk.

Influence of pyruvate on ammonia metabolism by renal cortical mitochondria.

The effect of the tricarboxylic acid (TCA) cycle precursor, pyruvate, on glutamine metabolism by isolated renal cortical mitochondria was assessed by quantitating its key nitrogen and carbon metabolites. When mitochondria from normal rats were incubated at pH 7.4, pyruvate (2 mM) inhibited ammonia production by almost completely erradicating glutamate deamination and by diminishing glutamine deamidation but to a lesser extent. Alpha KG, citrate, and malate accumulation in the incubation medium were increased dramatically reflecting the increased flux of pyruvate through the TCA cycle; the intramitochondrial concentrations of both Alpha KG and glutamate were increased. Thus, pyruvate primarily inhibits flux through glutamate dehydrogenase as a result either of an increase in Alpha KG concentration and/or a decrease in the redox (NAD/NADH) potential secondary to enhanced flux through the TCA cycle. Glutamine deamidation is secondarily inhibited, presumably due to the increased intramitochondrial concentration of glutamate. Citrate (2 mM) produced changes comparable to those observed with pyruvate. Mitochondria from normal rats incubated at pH 7.0 as well as mitochondria from rats with chronic metabolic acidosis responded to pyruvate in a fashion qualitatively similar to normal mitochondria incubated at pH 7.4. Glutamate deamination was inhibited significantly, but a high rate persisted with chronic acidosis despite the presence of pyruvate. Nevertheless, when glutamine metabolism was contrasted with normal mitochondria incubated at pH 7.4, the response to in vitro incubation in an acid pH as well as to chronic metabolic acidosis was similar quantitatively regardless of whether glutamine alone or in combination with pyruvate was present in the incubation medium.

Influence of diet composition on serum triiodothyronine (T3) concentration, hepatic mitochondrial metabolism and shuttle system activity in rats.

Two experiments were conducted to determine if variations in diet composition sufficient to alter circulating triiodothyronine (T3) concentration would influence hepatic mitochondrial metabolism. In experiment 1, mitochondrial respiration and the activity of succinate dehydrogenase (SDH), cytochrome oxidase (CO) and alpha glycerophosphate dehydrogenase (m alpha-GPD) were measured in 42-day-old male rats fed diets containing casein/carbohydrate/fat: 8/73/10% (low protein), 22/59/10% (control protein), and 45/36/10% (high protein) for 3 weeks. When compared to control, serum T3 was increased 2-3 times in the low and decreased 19% in the high protein-fed groups. Mitochondria isolated from low protein-fed rats consumed less oxygen in both state 4 and state 3 with succinate as substrate when compared to control or high protein fed rats. However, ADP/O and respiratory control (RC) ratios were similar in all groups. Activity of SDH and CO was decreased only in low protein-fed rats. M alpha-GPD activity was increased in the low and decreased in the high protein fed-rats. In experiment 2, alpha-glycerophosphate shuttle activity was increased 2-3 fold and malate-aspartate shuttle activity decreased 60% in intact mitochondria isolated from low protein-fed rats when compared to rats pair-fed control diet. These results suggest a role for diet composition as a regulator of hepatic intermediary metabolism mediated by thyroid hormones.

Effect of pH on metabolism of alpha-ketoglutarate by renal cortical mitochondria.

The effect of acid-base perturbations on mitochondrial alpha-ketoglutarate (alpha-KG) metabolism was quantitated by measuring the nitrogen and carbon metabolites of glutamine. alpha-KG metabolized was calculated as the difference between alpha-KG production from glutamine (glutamate deamination plus transamination) and alpha-KG accumulation in the medium. Under all experimental conditions accumulation in the medium of malate plus aspartate was altered similarly to the calculated change in alpha-KG metabolism. Mitochondria from rats with chronic acidosis were compared to pair-fed controls. Chronic acidosis resulted in increased alpha-KG production and its intramitochondrial concentration; the rate of conversion of alpha-KG to succinate was unchanged. When mitochondria from normal animals were incubated at pH 7.0, 7.4, and 7.7, the amount of alpha-KG metabolized was altered, but the magnitude and direction of the response was dependent on the concentration of glutamine (0.5, 1.0, or 5.0 mM). A low pH depressed production but stimulated the subsequent metabolism of alpha-KG, whereas an alkaline pH acted in the opposite fashion. The overall response at a given glutamine concentration depended on which effect predominated. Accordingly, chronic acidosis does not induce adaptive changes, but pH, per se, directly alters intramitochondrial alpha-KG metabolism.

Effect of hypophysectomy on calcium transport by rat duodenum.

To examine the effect of hypophysectomy on intestinal calcium absorption, studies were performed on immature rats 7, 14, and 21 days after hypophysectomy. Duodenal calcium transport was measured in vitro utilizing everted gut sacs and in vivo by a luminal perfusion technique. Hypophysectomy produced no differences in the ability of everted gut sacs to transport calcium. Similarly, when in vivo transport data were expressed on the basis of intestinal length, no significant differences were noted. However, when transport data were expressed on the basis of mucosal weight, increases in absorption and lumen-to-plasma fluxes were apparent in hypophysectomized animals. No differences were seen in plasma-to-lumen fluxes. The results indicate that when the transport data are corrected for mass of intestinal mucosa, the duodenum from hypophysectomized animals absorbs calcium more avidly due to an increase in lumen-to-plasma flux.

Effect of pH on ammonia production by renal mitochondria.

NH3 production by renal cortical mitochondria was studied under conditions of metabolic acidosis induced in vivo and with pH manipulations of the media bathing mitochondria from normal rats. A HCO3- medium equilibrated with O2 and CO2 was utilized with glutamine concentrations of either 10 or 0.5 mM. With chronic acidosis NH3 production increased significantly at either substrate concentration. Similar results were obtained with rotenone in the media, both with chronic acidosis and with acidosis of 3 h duration, indicating that increased glutamine entry and/or phosphate-dependent glutaminase (PDG) activity accounts for the increased ammoniagenesis. In contrast to acidosis induced in vivo, mitochondria from normal rats subjected to a diminution in medium pH, either by manipulation of HCO3 concentration or PCO2, significantly decrease NH3 production. Mitochondrial studies with rotenone, as well as studies of solubilized PDG, suggest that a low pH diminishes NH3 production by directly altering PDG activity. Furthermore, regardless of the specifics of the mechanism, these studies indicate that adaptation to metabolic acidosis is not the immediate, direct result of a change in pH.

Intestinal mitochondrial calcium uptake during adaptation to dietary calcium restriction.

In order to evaluate the role of mitochondrial calcium uptake in intestinal calcium absorption, the effect of adaptation to dietary calcium deficiency on the in vitro uptake of calcium by isolated duodenal mitochondria was studied. Experiments were performed utilizing 28-day-old cockerels which had received a diet adequate in vitamin D3 and phosphate and containing either 0.08% or 1.20% calcium. Mitochondrial 45Ca uptake from chicks deprived of dietary calcium was not significantly different from controls. These results suggest that increased calcium uptake by intestinal mitochondria is not crucial for adaptation to a low calcium diet.

Effect of potassium on ammoniagenesis by renal mitochondria.

Ammonia production by rat renal cortical mitochondria was studied with both in vivo and in vitro manipulation of potassium to further elucidate the mechanisms relating potassium homeostasis and renal ammonia production. Mitochondria from potassium-depleted animals demonstrated an increase in ammonia production at all glutamine concentrations studied, which ranged from 0.5 to 10 mM. This increase in ammoniagenesis compared favorably in degree of change with and was of sufficient absolute magnitude to entirely account for the findings observed both in renal cortical slices studied in vitro and in the intact animal. When rotenone is added to the medium, increased ammoniagenesis is still detected, indicating that either glutamine entry into the mitochondria and/or the activity of phosphate-dependent glutaminase are critical rate-controlling steps. In contrast to studies with renal slices, a decrease in ammonia production was not apparent with cortical mitochondria from chronically potassium-loaded animals. In vitro alterations of the potassium homeostasis. Therefore, these experimental manipulations either do not activate an effector mechanism which takes place in vivo, or the alteration in ammoniagenesis requires an adaptation over time that is not achieved with this in vitro approach.

The comparative effects of vitamin d deficiency and ethane-1-hydroxy-1,1-diphosphonate administration on the histology and glycolysis of chick epiphyseal and articular cartilage.

A comparison has been made between the effect of a vitamin D--deficient diet and treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the morphology of chick epiphyseal cartilage and on the production of lactate in vitro by epiphyseal and articular cartilage. The cell populations in the growth plate were different following the two treatments. Vitamin D deficiency was characterized by an increase in proliferating cells, with a relative paucity of hypertrophic cells; EHDP treatment was characterized by an increase in hypertrophic cells. When similar cell types were compared, neither treatment changed lactate production from the control value. This stresses the need to correlate the morphology of cell types with their metabolic function. The present results indicate that the major effect of vitamin D deficiency in the chick is to block the differentiation of proliferating to hypertrophic cells. In contrast, EHDP may act by inhibiting calcification directly. Even though EHDP at the doses used is known to interfere with the production of 1,25-dihydroxycholecalciferol there is no block to cell differentiation under EHDP similar to that seen in dietary deficiency of vitamin D.