Zinc nutritional status modifies renal osteodystrophy in uremic rats.

BACKGROUND: Previous studies from our laboratories suggested that zinc depletion reduces the circulating level of 1,25-dihydroxycholecalciferol (1,25(OH)2D, calcitriol) in calcium- and phosphorus-depleted rats with normal renal function, and rats with uremia. Since calcitriol synthesis is in part dependent on renal function, we studied levels of circulating vitamin D metabolites, PTH response, mineral balance and bone histomorphometry in animals with different zinc nutritional and renal functional status. METHODS: Fifty-eight male Sprague-Dawley rats were pair-fed zinc-replete (+) or -deplete (-) diets for two weeks. Thereafter, half of each paired group underwent nephrectomy (N), while half had sham (S) operations. Animals were observed for eight weeks after surgery. External mineral balances of zinc, calcium, phosphate and magnesium were determined before surgery, and 1, 2 and 7 weeks after surgery. Plasma creatinine, zinc, calcium, phosphorus, magnesium, 25-hydroxycholecalciferol, calcitriol and PTH were determined at sacrifice. Static and dynamic bone histomorphometry was determined by standard techniques. RESULTS: After an 8-week observation period, zinc-depleted animals had lower plasma zinc levels, and nephrectomized animals had lower creatinine clearances than respective controls at sacrifice. Plasma calcium and phosphorus concentrations were similar in all four groups at sacrifice. Plasma magnesium concentrations were similar in groups with renal insufficiency, regardless of zinc nutritional status. Plasma 25-hydroxycholecalciferol and calcitriol levels were similar in all groups. There was no difference between mean PTH concentration in sham-operated animals, regardless of zinc nutritional status. Although nephrectomized groups' PTH levels were increased compared to S controls, PTH levels were increased in +Zn/N animals compared to the -Zn/N group. Zinc-deplete groups had consistent negative net zinc balance, however, there was no consistent effect of nephrectomy on external calcium, phosphorus, or magnesium balance, when nephrectomized groups of different zinc nutritional status were compared. Nephrectomized animals had histomorphometric changes indicative of higher bone turnover and abnormal mineralization. Zinc deficiency was associated with less evidence of increased parathyroid hormone activity on bone in nephrectomized rats. CONCLUSIONS: Zinc depletion limits the increase in plasma PTH concentration and the expression of secondary hyperparathyroid bone disease during the development of renal insufficiency in the renal ablation model of uremia in rats. The mechanism underlying this effect is unknown, but may involve a direct effect of zinc on the synthesis, release, metabolic clearance, and/or action of PTH on the cellular level, on the interrelationship of calcitriol and PTH, or a direct effect of zinc on bone mineral metabolism. These data highlight the potential relevance of zinc nutritional status to mineral metabolism in patients with chronic renal insufficiency and end-stage renal disease.

Zinc nutritional status modulates the response of 1,25-dihydroxycholecalciferol to calcium depletion in rats.

Male Lewis rats (n = 27) were fed a nonpurified diet containing 0.9% calcium, 0.7% phosphorus, and 0.005% zinc until 8 wk of age. At this time rats were assigned randomly to one of two groups. Both groups were fed a low calcium, low zinc, purified diet (0.2% calcium, 0.4% phosphorus, less than 0.00007% zinc), but one group was fed 1.78 mg Zn/(animal.d). The zinc-replete animals were individually matched by weight to the zinc-depleted animals and pari-fed. Balances and plasma concentrations of zinc, calcium, and phosphorus and parathyroid hormone, 25 hydroxycholecalciferol [25(OH)D] and 1,25-dihydroxycholecalciferol [1,25(OH)2D] were determined at the start of calcium depletion and 2 wk later. Calcium and 25(OH)D levels were lower in both groups after calcium depletion. Dietary zinc had no significant effect on calcium or 25(OH)D levels. Phosphorus concentrations were lower after calcium depletion, but phosphorus concentration was higher in the zinc-depleted compared with the zinc-replete group at the end of the experiment. 1,25(OH)2D increased in both groups, but was higher in the zinc-replete than the zinc-depleted group at the end of the experiment. Calcium and phosphorus balances were greater in the zinc-depleted group at the end of the experiment. We conclude zinc depletion diminishes the response of 1,25(OH)2D to calcium depletion in rats. The mechanism is unknown, but may involve nonhormonally mediated changes in gastrointestinal absorption of calcium and phosphorus or an affect of zinc on extraintestinal processes.

Magnesium and calcium absorption in Fischer-344 rats influenced by changes in dietary fibre (wheat bran), fat and calcium.

Magnesium and calcium absorption were affected by changes in dietary wheat bran fibre and calcium, but not fat, in Fischer-344 rats when studied in a full factorial study which was a portion of a larger study of diet and colon carcinogenesis. For four weeks, nine-week-old rats were fed experimental purified diets to which had been added: wheat bran 0, 2.5 10, or 20%; fat 1, 5 or 10%; and calcium 0.18, 0.52, or 1.04% of diet weight. From day 26 to 29 all faeces were collected in metabolic cages, and food consumption noted. Dietary magnesium intake and net magnesium absorption increased in direct relation to the quantity of wheat bran in the diet. Calcium supplementation inhibited magnesium absorption on fibre-free diet, but had little effect on magnesium absorption when fibre was present. Fat had no measurable effect on magnesium absorption. A low dietary fibre content enhanced Ca absorption compared to that on a fibre-free diet. However, further increases in fibre content slightly inhibited calcium absorption. We conclude that the magnesium content of dietary wheat bran fibre is available for absorption to rats. Calcium supplementation inhibits magnesium absorption in a fibre-free diet, but presence of dietary fibre protects magnesium absorption from the calcium inhibition observed on a fibre-free diet. Absorption of calcium is increased by including some fibre in the diet. However, calcium absorption may be diminished slightly by increasing wheat bran content of the diet to a high level, probably through calcium binding and excretion with undigested fibre.

Zinc binding in intestinal brush-border membrane isolated from pig.

Zinc binding to brush-border membrane vesicles isolated from pig jejunum was investigated by a rapid filtration method, for long incubation periods (up to 180 min). Zn2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160 microliters/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 microliters/mg protein). Changes in medium osmolarity had no effect on zinc accumulation. These results suggested a large degree of zinc binding to vesicular components (membrane or core). 65Zn efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competitive cations, and a large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with an exchange molecule or a chelator. Scatchard plot analyses revealed, on one hand a first class of high-affinity extravesicular zinc binding sites (Ka = 8.6.10(3) M-1, n = 0.455 nmol Zn2+/mg protein) and a second class of extravesicular sites having a very low affinity (Ka = 22 M-1, n = 25.35 nmol Zn2+/mg protein) and, on the other hand one type of intravesicular sites (Ka = 3.3.10(4) M-1, n = 550 nmol Zn2+/mg protein). The intravesicular sites have a high affinity for zinc and are specific, since only nonlabelled zinc (or cadmium) but not calcium present in the bathing medium is exchanged with the internally accumulated labelled cation.

Zinc nutritional status modulates the 1,25-(OH)2D. Response in uremic rats.

Previous studies from our laboratories have suggested that zinc depletion reduces the circulating level of 1,25-dihydroxycholecalciferol-1,25-(OH)2D-in phosphorus-depleted rats. Since calcitriol synthesis is in part dependent on renal function, we studied vitamin D metabolites, parathyroid hormone response, and mineral balance in animals with different zinc nutritional and renal functional status. Twenty-three male Lewis rats were pair fed with zinc-replete or zinc-deplete diets for 2 weeks. Thereafter, half of each paired group underwent nephrectomy, while half had sham operations. After a 4-week observation period, the zinc-depleted animals had lower plasma zinc levels, and nephrectomized animals had higher plasma creatinine concentrations than respective controls at sacrifice. Plasma calcium and phosphorus concentrations were similar in all four groups at sacrifice. The 25-hydroxycholecalciferol and parathyroid hormone concentrations were similar in groups with renal insufficiency, regardless of the zinc nutritional status. The mean plasma 1,25-(OH)2D concentration was 20 +/- 4 pg/ml in the zinc-replete/sham-control group. In the zinc-replete nephrectomized animals, the mean plasma 1,25-(OH)2D concentration was increased by 133% to 56 +/- 6 pg/ml, as compared with zinc-deplete nephrectomized group. There was a significant effect of renal function, zinc nutritional status, and the interaction of these factors in accounting for differences in mean 1,25-(OH)2D levels. Zinc-deplete groups had consistent negative net zinc balance. However, there was no consistent effect of zinc nutritional status on external calcium or phosphorus balance when nephrectomized groups of different zinc nutritional status were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25(OH)2D response to combined zinc and phosphorus depletion in rats.

To investigate the role of zinc depletion in the response of calcitriol to a single provocative stimulus, we studied vitamin D metabolites, parathyroid hormone (PTH), and mineral balance during phosphorus depletion in animals with different zinc nutritional status. Male Lewis rats (66) were pair fed normal phosphorus (NP), zinc-replete (+) or -deplete (-) diets for 2 wk. Thereafter, one-half of each paired group underwent 1 wk of phosphorus depletion (LP). Zinc-deplete animals had lower plasma zinc, and phosphorus-deplete animals had lower plasma phosphorus concentrations than respective nutritionally replete controls. Plasma calcium, PTH, and 25-hydroxycholecalciferol concentrations were similar in all four groups at end of experiment. Mean plasma 1,25(OH)2D concentration increased 80% from 35 +/- 3 to 63 +/- 9 pg/ml (P less than 0.007) when values were compared before and after phosphorus restriction in zinc-replete animals (LP + group). The 7.9% increase from 35 +/- 4 to 38 +/- 4 pg/ml in 1,25(OH)2D concentration in animals with combined zinc and phosphorus depletion (LP-) was not significant. External calcium balance, however, was maintained in the combined zinc and phosphorus-deplete group. We conclude that zinc depletion limits the increase in plasma 1,25(OH)2D concentration associated with phosphorus depletion. The mechanism is unknown but may involve an effect of zinc on renal 25(OH)D 1-alpha-hydroxylase synthetic activity.

Studies of zinc transport into brush-border membrane vesicles isolated from pig small intestine.

Zinc transport into brush-border membrane vesicles was investigated by measuring uptake rates at a very short incubation time (2 seconds), during the initial linear uptake. A divalent cation chelator (EGTA) was added to the stop and washout solutions in order to remove the zinc bound to the external surface of the vesicles. Under these conditions, we showed that zinc enters the vesicles by (1) a saturable carrier-mediated process, and (2) an unsaturable pathway. The kinetic parameters we calculated were an affinity of 0.215 +/- 0.039 mM, a Jmax of 17.2 +/- 1.7 nmol.min-1.(mg protein)-1 and an unsaturable constant of 0.025 +/- 0.006 (n = 6). The imposition of an outwardly directed K+ gradient (negative inside) did not affect the Jmax value of the zinc uptake but increased the Km value significantly. This suggests that, at least a portion of zinc which crosses the membrane does not do so in a cationic form. Zinc uptake was decreased or increased according to the nature of accompanying anions (Cl-, SO4(2)-, SCN-) in the absence of any membrane potential. With highly permeant anions such as thiocyanates, zinc uptake was considerably augmented, suggesting a movement of zinc in a complexed form involving the presence of negative species. We also showed that cadmium competitively inhibited the zinc uptake; we measured a Ki value of 0.21 mM, indicating a similar affinity of cadmium for the carrier as zinc itself. By contrast, the presence of calcium had little effect on zinc entry into vesicles. The calcium ionophore A23187 had only a slight stimulating effect on zinc uptake. These results indicate that zinc and calcium transports are probably independent of each other.

Zinc inhibition of glucose uptake in brush border membrane vesicles from pig small intestine.

The effect of zinc on sodium coupled glucose uptake was studied in pig intestinal brush border membrane vesicles. In this system zinc inhibited glucose uptake and appeared to have a Ki of 0.25 mM. When tested by spectrophotometry, electron microscopy and protein determination following centrifugation, no evidence of significant vesicle aggregation was found with 0.5 mM zinc treatment. Zinc inhibition of glucose uptake persisted when the vesicle membrane potential was clamped with identical KCl concentrations inside and outside the vesicles in the presence of valinomycin. Variation of the glucose and sodium concentrations gave results indicating that zinc reduces glucose affinity for the carrier but not sodium binding to the transporter. The glucose inhibitory effect was not due to a rapid dissipation of the sodium gradient as zinc failed to affect sodium uptake in the absence of glucose. Zinc also failed to inhibit glucose efflux from vesicles under isotopic exchange conditions, when glucose and sodium concentrations were identical inside and outside vesicles. The t1/2 of glucose inhibition by zinc was relatively long, i.e. 6 min. We conclude that zinc acts as an inhibitor of glucose transport by interacting with the sodium-glucose co-transporter. The long zinc incubation time required to achieve maximal inhibition of glucose transport suggests that this interaction takes place within vesicles.

Zinc balance in combined zinc deficiency and uremia.

In order to evaluate whether the hypozincemia associated with chronic renal failure represents true negative zinc balance, male Lewis rats were randomized and pair-fed zinc replete (+Zn) or deplete (-Zn) diets. Subsequently, each animal underwent one- and two-thirds nephrectomy, or a sham operation. Complete balances for zinc, calcium, and magnesium were determined weekly before and after nephrectomy. At sacrifice, -Zn animals had significantly lower (P less than 0.005) plasma zinc levels compared to controls. Nephrectomized (N) animals had significantly elevated plasma creatinine (P less than 0.005), diminished creatinine clearances (P less than 0.005), and increased urinary flows (P less than 0.005) and protein excretion (P less than 0.01) compared to sham-operated (S) controls. Zinc nutritional status had no effect on these parameters in animals with comparable renal function. -Zn/N animals had consistently significantly elevated (P less than 0.005) urinary zinc excretion compared with -Zn/S with similar plasma zinc concentrations. Urinary zinc excretion correlated with urinary flow (r = 0.739, P less than 0.005), urinary protein (r = 0.635, P less than 0.01) and urinary calcium excretion (r = 0.855, P less than 0.005) in -Zn groups. -Zn/N rats had a significantly larger fecal zinc excretion for the first week post-nephrectomy, compared to -Zn/N controls (P less than 0.005). This resulted in negative zinc balance in -Zn/N animals, compared to their pre-nephrectomy balance (P less than 0.05) or to -Zn/S controls for the first (P less than 0.005) and second (P less than 0.05) weeks. -Zn/N rats had lower (P less than 0.02) hepatic zinc concentrations compared to the -Zn/S group.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary fiber supplementation and fecal bile acids, neutral steroids and divalent cations in rats.

Young adult rats were fed ad libitum for 4 wk on defined diets containing either no fiber, 10% levels of insoluble fiber sources [cellulose, wheat bran, alfalfa, mixed fibers (Fibyrax)], 5% levels of soluble fiber sources [pectin, guar gum, psyllium (Metamucil)] or 2% cholestyramine (Questran), a bile acid sequestrant. Fecal samples were obtained from paired rats over a 3-d period, were analyzed for neutral and acidic steroid levels and compositions and, together with the food, were assayed for divalent cations (Ca2+, Mg2+, Fe2+ and Zn2+). Animals in all groups were in balance for divalent cations, and there appeared to be no relationship between the extent of cation balance and the type of fiber fed. All insoluble fiber diets and the guar gum and psyllium diets resulted in significantly higher daily fecal mass and, in general, resulted in significant dilution of total fecal steroids. With the insoluble fibers, there was a general dilution of fecal neutral steroids, which was not apparent with the soluble fibers or cholestyramine. In addition, except for the pectin- and mixed fiber-containing diets, there was reduced bacterial conversion of the primary bile acids to secondary bile acids or metabolites.

Alterations in calcium, magnesium, iron, and zinc metabolism by dietary cholestyramine.

Cholestyramine is an effective drug for the reduction of plasma cholesterol because of its ability to sequester intestinal bile acids. Since metabolic alterations, including diminished intestinal absorption of vitamin D and osteomalacia have been reported with long-term use of this resin, the influence of cholestyramine on dietary balance of four mineral elements has been investigated. Wistar-strain rats were fed either a 2% cholestyramine or control diet for one month. Dietary intakes and fecal and urinary excretions of calcium, magnesium, iron, and zinc were determined using atomic absorption spectrophotometry during three, 3-day balance periods. Cholestyramine-fed rats had a net negative balance for calcium and a lower net positive balance for magnesium, iron, and zinc than the controls. Other effects of cholestyramine were an increased urinary excretion of calcium and magnesium, a decreased urinary zinc, and an alkalinization of urine. Blood and tissue cation content was unchanged except for a reduction in serum magnesium with resin feeding. Alterations in calcium, magnesium, and zinc metabolism might be explained by inadequate vitamin D absorption from the intestine followed by an increased secretion of parathyroid hormone. A diminished iron absorption due to resin binding could account for the reported disturbance in iron balance.

Urinary zinc in relation to other cations and flow during volume expansion and intravenous chlorothiazide.

Urinary excretion of zinc, sodium, potassium, and calcium was studied in anesthetized dogs under conditions of volume expansion by saline infusion and volume expansion plus chlorothiazide administration. Zinc excretion was positively correlated to the fractional water excretion, as well as to th excretion of the other cations, during volume expansion. Chlorothiazide administration during volume expansion increased the zinc, sodium, and potassium excretion without changing that of calcium. The enhanced zinc excretion during chlorothiazide diuresis was equal to that expected on the basis of the increase in fractional water excretion alone. The urinary concentration of zinc appeared inversely related to the urine flow rate, reaching a minimum below that of the plasma ultrafilterable zinc concentration. The ratio of the clearance of zinc to that of sodium was 0.28, indicating a greater degree of net reabsorption for zinc than for calcium. These findings suggest that zinc and sodium reabsorption may be inhibited to a similar degree at chlorothiazide-sensitive sites in the tubule. Furthermore, the zinc reabsorptive mechanism seems capable of lowering urinary zinc concentration below that of ultrafiltrate and appears related in some way to sodium reabsorption.

Luminal alkalinization by guinea-pig cecum in vitro, an electro-neutral process.

Guinea-pig cecum was found to alkalinize its mucosal media in vitro at a chemical equivalents rate greater than the short circuit current (ISC). Alkalinization was inhibited by conditions which did not affect ISC and by low mucosal Na suggesting an electro--neutral process, dependent on Na.

Receptive-field properties of neurons in binocular and monocular segments of striate cortex in cats raised with binocular lid suture.

1. We studied the receptive fields of 171 striate cortical neurons from 17 cats raised with binocular lid suture. Of these, 102 fields were within 10 degrees of the area centralis and the remaining 69 were at least 38 degrees from the vertical meridian. 2. Based on their different response properties, cells were divided into three broad groups: the mappable cells (49%) had clearly defined receptive fields, the unmappable cells (31%) were activated by visual stimuli but had diffuse fields which could not be hand plotted, and the visually inexcitable cells (20%) could not be activated by visual stimuli. Very few (less than or equal to 12% of the total sample) normal simple or complex cells could be found. 3. Orientation selectivity was assessed in these cells. Only 12% displayed orientation selectivity within normal bounds, and these were all mappable cells. None of the unmappable cells had discernible orientation selectivity. 4. Ocular dominance was assessed for 62 of the centrally located receptive fields. Among mappable cells, there was an abnormally low proportion of binocular fields, while no such abnormality was seen for unmappable cells. 5. For 47 of the neurons, average response histograms were compiled for moving stimuli of various parameters in an effort to evoke the maximum discharge or peak response. This peak response was normal for mappable cells but reduced for unmappable cells. 6. We devised a technique for studying potential inhibitory receptive-field zones in these neurons, validated the method in normal striate cortex, and used it to test 20 mappable cells in the lid-sutured cats. None showed the pattern of strong inhibitory side bands seen in normal simple cells, although six showed weak or abnormal inhibitory zones. Interestingly, six of the seven visually inexcitable cells tested by this method had purely inhibitory receptive fields. 7. The effects of binocular suture were essentially identical for the binocular and monocular segments since the cell types and their response properties did not differ between these two areas of cortex. Furthermore, the cortical monocular segments of these cats seemed qualitatively different from the deprived cortical monocular segment after monocular suture. This extends an analogous difference for these cats reported for the monocular segments of the lateral geniculate nucleus. We thus conclude that monocularly and binocularly sutured cats develop by qualitatively different mechanisms. For the former, competition between central synapses related to each eye is a prominent feature of geniculocortical development, whereas, for the latter, such specific forms of geniculocortical development may not obtain.

Laser Doppler measurement of cutaneous blood flow.

This work describes an instrument for the noninvasive measurement of cutaneous blood flow velocity. The system utilizes the Doppler shift of laser light backscattered from moving red blood cells in the cutaneous microcirculation, the shift being obtained by an optical heterodyning technique. Comparison is made between this technique and the 133xenon clearance technique in measuring cutaneous flow in the forearms of normal volunteers. Variations in flow were obtained by inducing different degrees of solar erythema with an ultraviolet sunlamp. A Y on X linear regression yielded a regression coefficient = 0.89 (p less than 0.001, n = 16) between the two methods. The laser Doppler method appear to represent a practical technique for clinical evaluation of cutaneous blood flow in any skin surface.

An anatomical study of some somesthetic afferents to the intercollicular terminal zone of the midbrain of the opossum.

Following lesions of the dorsal column nuclei, spinal cord and sensorimotor cortex, anterograde degeneration was traced to a common target in the central midbrain; the intercollicular terminal zone. Although not all the midbrain projections of the three pathways are contained within this zone, it receives overlapping projections from each of the three body-related somesthetic pathways studied. The intercollicular terminal zone covers the entirety of the external nucleus of the inferior colliculus and the intercollicular nucleus of Mehler, spilling over into adjacent parts of the central gray and deep layers of the superior colliculus. Little evidence of somatotopy in the somesthetic projections was disclosed, and bilateral input was found in each experimental case, regardless of the type of lesion. Golgi-stained material was examined to explore the basis of the extensiveness of the terminal zone. These materials showed that the dendritic spread of neurons in the external nucleus of the inferior colliculus and the intercollicular nucleus, taken together, covers the entirety of the intercollicular terminal zone. That is, the dendrites invade the same portions of the central gray and deep layers of the superior colliculus which are covered by the terminal field. We conclude that a tripartite division of the sensory midbrain may be justified: with the somesthetic modality being represented anatomically in a fashion at least roughly analogous to that of vision and audition.