Lipid accumulation in jejunal and colonic mucosa following chronic cholestyramine (Questran) feeding

The hypolipidemic agent, cholestyramine (Questran), when fed to rats inhibits intestinal absorption of cholesterol and triglycerides and causes significant epithelial cell damage in both small and large intestine. In this study, we report significant accumulation of lipids in the mucosal layer of both jejunum and colon in rats administered 2% cholestyramine for a four-week period, when compared to a control group maintained on regular chow. The total lipid increment with cholestyramine was 4.7-fold in the jejunum and 3.7-fold in the colon. The triglyceride fraction increased substantially in the small but not the large intestine. Relative phospholipid levels decreased in the treated jejunum but not in the colon. The biochemical data were reflected in morphological evidence of lipid-laden enterocytes obtained by light and transmission electron microscopy. Since cholestyramine has been shown to sequester 99.8% of micellar phospholipidin vitro, it is concluded that the presence of cholestyramine in the intestinal lumen may interefere with phospholipid availability for chylomicron synthesis and serosal lipid exit from the epithelium. This unusual deposition of lipid within the mucosal layer may also be correlated with the known cocarcinogenic effect of this resin in experimentally induced intestinal cancer.Supported by USDA grants 82CRCR 1071 and 82 CRCR 1001.     

Treatment with high-dose oral vitamin D2 in patients with jejunoileal bypass for morbid obesity. Effects on calcium and magnesium metabolism, vitamin D metabolites, and faecal lag time

Calcium and magnesium balance, 47Ca turnover studies, and measurements of vitamin D metabolites were performed before and after 7-10 months of vitamin D2 treatment (36,000 IU/day) in eight patients bypass-operated 3-6 years earlier for gross obesity. All patients had received a daily supplementation of calcium (27 mmol/day) since operation. Before treatment the net calcium absorption and calcium balance were normal compared with that of nine normal controls. Vitamin D metabolites were within normal limits. The endogenous faecal calcium level was increased and the faecal lag time shortened. Bone biopsies revealed osteomalacia in three of the patients. Vitamin D2 treatment induced an increase in calcium absorption and renal excretion of calcium, a reduced bone resorption rate, a more positive calcium balance, and healing of osteomalacia. Moreover, the vitamin D2 treatment induced a prolongation and normalization of faecal lag time, an increase in magnesium absorption, and a more positive magnesium balance. The effect might be mediated through 25-hydroxyvitamin D (25-OHD), which increased, whereas serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D) and 24,25-dihydroxyvitamin D (24,25-(OH)2D) were unchanged. The results indicate that in some bypass-operated patients high-dose vitamin D2 has a beneficial effect on calcium and magnesium metabolism.     

Hypercalciuria and altered intestinal calcium absorption occurring independently of vitamin D in incomplete distal renal tubular acidosis

Negative calcium balance and calcium nephrolithiasis are two sequelae of chronic metabolic acidosis. To establish the effects of acidosis on calcium and vitamin D metabolism, we have examined a group of nine patients with incomplete distal renal tubular acidosis. Patients were studied during a control phase and after eight months of potassium citrate treatment, 60 to 80 meq daily. Potassium citrate caused a significant decrease in urinary calcium. The fractional intestinal calcium absorption increased significantly, yet no change was observed in serum vitamin D levels. The estimated calcium balance increased significantly from -70.2 +/- 63.8 to +66.7 +/- 48.7 mg/d (P less than 0.01). Thus, potassium citrate treatment improved the estimated calcium balance by lowering urinary calcium while increasing the fractional intestinal calcium absorption. The original hypercalciuric state, its correction to normocalciuria, and the augmentation of intestinal calcium absorption seen in these patients are probably independent of vitamin D control since there was no change noted in serum 1,25-dihydroxyvitamin D levels.     

Effect of cholestyramine on the absorption of vitamin D3 and calcium

The absorption of 10 mug doses of vitamin D(3)-(3)H in rats was found to be decreased by the addition of cholestyramine to the diet in an amount sufficient to cause steatorrhoea. Studies in vitro suggested that the effect of cholestyramine was due to its known ability to bind bile salts and thus disrupt micelles containing vitamin D.The absorption of 1 mg (47)calcium chloride was similar in both control and cholestyramine-fed rats, whether estimated by the whole body counting or the faecal excretion technique.These findings show that the administration of cholestyramine causes a reduction in the absorption of vitamin D(3)-(3)H but not of radiocalcium.     

Pathogenesis of hyperoxalurias and calcium oxalate calculi in intestinal diseases

Hyperoxaluria and calcium oxalate lithiasis have a multifactorial genesis in bowel diseases. The augmented synthesis of oxalic acid in the liver is of minor importance. A diminished bacterial degradation is to asses up to day only hardly. The increasing absorption is of highest importance by deficient production of calcium oxalate in the intestinal tract and by increasing permeability of colonic mucosa above all in steatorrhoea or in patients with augmented calcium absorption. The crystallization of calcium oxalate in the urine is promoted by shortage of vitamin A, citrate, zinc or magnesium.     

Effects of dietary sodium chloride loading on parathyroid function, 1,25-dihydroxyvitamin D, calcium balance, and bone metabolism in female rats during chronic prednisolone administration

The effects of dietary sodium chloride supplements (8 g/100 g diet) on parathyroid function, serum 1,25-dihydroxyvitamin D [1,25-(OH)2D], calcium balance, bone metabolism, and bone composition were studied in rats treated with prednisolone (2 mg/kg w X day) for 12 weeks. Animals on a low calcium diet (0.1% Ca) received the following treatments: group 1, control; group 2, NaCl; group 3, prednisolone; group 4, NaCl plus prednisolone. Parathyroid function was assessed indirectly from urinary cAMP excretion: bone resorption was estimated by studying urinary hydroxyproline excretion and mobilization of 45Ca from bone. Dietary salt loading increased the urinary excretion of calcium, 45Ca, cAMP, and hydroxyproline and raised serum 1,25-(OH)2D and net calcium absorption, but lowered calcium retention, femoral calcium, and total body calcium. Prednisolone slowed body growth and lowered net calcium absorption, calcium retention, femoral calcium, and total body calcium. Urinary calcium excretion was higher in rats receiving salt and prednisolone in combination than in animals taking salt without prednisolone, but other responses to salt and prednisolone were independent. Thus, salt and prednisolone each elicit osteopenia, and salt causes bone loss in rats receiving prednisolone. The osteopenic effect of salt is attributed to primary augmentation of urinary calcium excretion and secondary increases in PTH-medicated bone resorption. Although salt-treated rats have higher blood levels of 1,25-(OH)2D, bone loss occurs because alimentary calcium absorption is not elevated sufficiently to offset urinary calcium losses. Prednisolone lowers bone formation and net calcium absorption without lowering serum 1,25-(OH)2D values. The parathyroid-vitamin D axis remains intact in prednisolone-treated rats, as they show increases in PTH and 1,25-(OH)2D after salt treatment.     

Intestinal calcium absorption is enhanced by D-glucose in diabetic and control rats

The effect of glucose on intestinal absorption of calcium was studied in the jejunum and ileum of control, diabetic (streptozotocin-induced), and insulin-treated diabetic rats. Intestinal absorption was determined in vivo using an in situ one-pass perfusion technique. In the jejunum of control and diabetic rats, net absorption and unidirectional lumen to mucosa flux of calcium and net absorption of water were significantly greater during perfusion of an isotonic NaCl solution, containing 15 mmol/L of glucose, than during perfusion of the same solution containing 15 mmol/L of mannitol instead of glucose. To determine if net absorption of calcium and water were related, the jejunum was perfused with a hypotonic solution (260 mosmol/kg) in separate groups of rats. Although net absorption of water was equivalent during perfusion of the hypotonic solution to that noted during perfusion of the isotonic glucose-containing solution, rate of absorption of calcium was not enhanced. Thus, it appeared that, if the enhancement in absorption of calcium by glucose was an effect on the passive absorption of calcium, it was through a mechanism not related to passive absorption of water. As expected, jejunal absorption of calcium was lower in diabetic than in control rats. Because in diabetic rats the metabolism of vitamin D to its active metabolite, 1,25-dihydroxy vitamin D, is defective, the enhancement in absorption of calcium by glucose did not appear to be due to a mechanism influenced by vitamin D metabolism. In the ileum, rate of absorption of calcium was lower than in the corresponding jejunum and was not significantly altered by the presence of glucose in the perfusion solution, by perfusion of a hypotonic solution, or by the diabetic state. The mechanism of action of glucose on calcium absorption in the jejunum needs to be studied further.     

Recovery of rats from vitamin D-deficient mothers.

Two separate studies were conducted using weanling rats from either an unsupplemented, low vitamin D colony or a supplemented, adequate vitamin D colony. Severe hypocalcemia, slower increases in body weight gain, and lower apparent calcium, magnesium, and phosphorus balance values occurred in the rats from the low vitamin D colony fed a purified AIN-76A, vitamin D-devoid diet compared to rats from the vitamin D-adequate colony fed the same diet. Apparent calcium, magnesium, and phosphorus balance values, as well as most other measurements, in rats from the low vitamin D colony fed a purified AIN-76a, vitamin D-adequate diet were greater than or equal to those of rats never subjected to low vitamin D. This was suggestive of overcompensation in recovery from low maternal vitamin D. However, rats from the low vitamin D colony fed an unrefined (chow), vitamin D-adequate diet had lower apparent balance and bone values compared with rats from the vitamin D-supplemented colony fed the same diet. Presumably high levels of calcium, magnesium, and phosphorous in the unrefined diet prevented any overcompensation during recovery, as occurred with purified diets, from the earlier vitamin D deficiency. Overall, results indicated weanling rats from a low vitamin D colony had low vitamin D stores and were marginally vitamin D-deficient. In addition, recovery from the marginal deficiency had occurred to a large extent after feeding a purified, vitamin D-adequate diet. The results suggest the use of low vitamin D colony rats as a model for human, marginal vitamin D deficiency.     

Bone mineral homeostasis in spontaneously diabetic BB rats. I. Abnormal vitamin D metabolism and impaired active intestinal calcium absorption

Calcium homeostasis was investigated in male BB rats with a diabetes duration of 3-4 weeks and compared with that in nondiabetic littermates either fed ad libitum or receiving selective semistarvation or an oral Ca supplement to obtain additional weight-matched and Ca intake-matched control groups. Diabetic rats had markedly increased food and Ca intake, so that their net Ca balance remained positive despite a 13-fold increase in urinary Ca excretion and a disappearance of active duodenal Ca absorption. Decreased duodenal Ca uptake correlated with decreased 1,25-(OH)2D3 levels (89 +/- 15 vs. 160 +/- 13 pg/ml in nondiabetic rats), decreased duodenal 9K Ca-binding protein concentrations (10 +/- 1 vs. 21 +/- 2 micrograms/mg protein), and decreased number of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-binding sites in duodenum, although the binding affinity was above normal. Nondiabetic Ca-supplemented rats exhibited a similar response: decreased 1,25-(OH)2D3 (95 +/- 8 pg/ml) and 9K Ca-binding protein (7 +/- 0.5 micrograms/mg protein) concentrations, decreased active duodenal Ca uptake, increased urinary Ca excretion, and a normal net Ca balance. Plasma vitamin D-binding protein levels were decreased by 62% in diabetic rats, due to a marked decrease in production rate, while the plasma half-time remained normal. The free 1,25-(OH)2D3 index was highest in diabetic rats, suggesting partial vitamin D resistance at the duodenal level. In semistarved rats, 1,25-(OH)2D3 levels and active Ca uptake remained normal, and the free 1,25-(OH)2D3 index was increased, together with suppressed vitamin D-binding protein levels. These studies indicate that nutritional abnormalities may contribute to but cannot totally explain the disturbances in vitamin D metabolism, transport, or action at the intestinal level.     

Duodenal active transport of calcium and phosphate in vitamin D-deficient rats: effects of nephrectomy, Cestrum diurnum, and 1alpha,25-dihydroxyvitamin D3.

Both the methanol:chloroform extractable material from the leaves of the Solanaceous plant, Cestrum diurnum (C.d.), and a 270 ng dose of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) increased the active absorption of calcium and phosphate across the proximal duodenum, studied in vitro, from sham-operated and nephrectomized (NPX) vitamin D-deficient rats. In these studies, conducted 24 h after surgery, the uremic state in the NPX animals markedly diminished the intestinal transport response to 1alpha,25-(OH)2D3 and also lowered baseline transport values across duodenum from the NPX vitamin D-deficient controls. Both C.d. and 1alpha, 25-(OH)2D3 elevated plasma Ca levels equally well in the sham-operated and NPX groups. The stimulation of intestinal Ca absorption in NPX animals indicates that, like the leaves of the South American plant, Solanum glaucophyllum, C.d. contains materials which can function in an analogous manner to compounds in the vitamin D group that have either a 1alpha hydroxyl group or its steric equivalent.     

Renal Control of Calcium,Phosphate, and Magnesium Homeostasis

Calcium, phosphate, and magnesium are multivalent cations that are important for many biologic and cellular functions. The kidneys play a central role in the homeostasis of these ions. Gastrointestinal absorption is balanced by renal excretion. When body stores of these ions decline significantly, gastrointestinal absorption, bone resorption, and renal tubular reabsorption increase to normalize their levels. Renal regulation of these ions occurs through glomerular filtration and tubular reabsorption and/or secretion and is therefore an important determinant of plasma ion concentration. Under physiologic conditions, the whole body balance of calcium, phosphate, and magnesium is maintained by fine adjustments of urinary excretion to equal the net intake. This review discusses how calcium, phosphate, and magnesium are handled by the kidneys.     

Vitamin D metabolism and the response to 1,25-dihydroxycholecalciferol in Osteoporosis.

The metabolism of isotopically-labelled cholecalciferol and the response to small doses of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in a group of women with osteoporosis presenting with crush vertebral fracture. No abnormality of vitamin D metabolism was detected. The administration of 1 microgram 1,25-(OH)2D3 for between 8 and 20 days was associated with an increased intestinal absorption and urinary excretion of calcium but caused no improvement in calcium balance. There was a small but significant rise in serum calcium and phosphorus and significant reduction in immunoassayable parathyroid hormone levels during treatment. It is concluded that 1,25-(OH)2D3 is unlikely to be of value in the management of osteoporosis.     

Is vitamin D3 essential for mineral metabolism in the Damara mole-rat (Cryptomys damarensis)?

The Damara mole-rat (Cryptomys damarensis) leads a strictly subterranean existence in an extensive maze of plugged burrows and eats a strictly herbivorous diet. An obvious source of vitamin D3 (D3), an important hormone in mineral homeostasis, therefore is lacking in this mammal. The effects of orally ingested D3 on mineral balance were investigated. In the normal D' status group of mole-rats, the apparent fractional intestinal absorption of calcium (91.1%), magnesium (91.2%), and inorganic phosphorous (91.2%) was high and increased slightly, (calcium, 94.5%; magnesium, 93.2%; inorganic phosphorous, 92.9%) after D3 administration. The amount of element absorbed was positively correlated with the amount ingested (P less than 0.001), suggesting that absorption occurs via a nonsaturable process. This might be an adaptation to the sparseness of natural food. The observed positive retention of calcium (94%), magnesium (85%), and inorganic phosphorous (99.9%) is attributed to their evergrowing teeth. These teeth are constantly worn down during digging and it is speculated that they represent a mineral "sink," assisting in the maintenance of mineral homeostasis. It is also hypothesized that mole-rats have adapted to an environment devoid of D3 and can attain sufficient calcium for their needs via nonvitamin D3-dependent processes. Although mole-rats have the "machinery" to use D3 and D3 can affect mineral balance, this effect may be disadvantageous if invoked.     

Disorders of calcium and magnesium homeostasis

The components of calcium and magnesium balance and the factors responsible for the maintenance of the serum concentration of these cations are reviewed. Within this framework, the causes and treatment of disturbances of the serum concentration are discussed. Hypercalcemia is usually a reflection of increased bone resorption and/or gut absorption with the kidney playing a secondary role. Hypocalcemia is usually due to either a disturbance in the parathyroid hormone-adenylate cyclase system or a disturbance in vitamin D metabolism. As vitamin D is required for expression of the action of PTH at bone and as PTH is a prime regulator of vitamin D metabolism, the absence of either component results in important disturbances in calcium balance. In contrast to calcium homeostasis, the kidney plays a major role in the determination and regulation of serum magnesium. The major causes of hypermagnesemia therefore are associated with loss of renal function, and hypomagnesemia is frequently due to renal magnesium wasting.     

The apparent vitamin D resistance of chronic renal failure: A study of the physiology of vitamin D in man

Biologic assay of the antiricketic activity in serum and tissues has been used, with concomitant measurements of intestinal net absorption of calcium, to study aspects of vitamin D metabolism in man. Patients with chronic renal failure appear to require a greater intake of the vitamin and a higher serum antiricketic activity, to promote calcium absorption, than does the healthy subject. This increased requirement is not due to intestinal malabsorption of the vitamin nor, apparently, to its metabolic inactivation nor loss in the urine. It could be caused by relative unresponsiveness of the target organ; or, if due to an acquired abnormality of vitamin D metabolism, this is likely to involve some metabolic transformation subsequent to the initial hydroxylation.     

Effects of diabetes and vitamin D-depletion on duodenal and ileal calcium transport in the rat

Summary We compared effects of vitamin D depletion and alloxan-induced diabetes on intestinal calcium transport in growing rats. Transport of⁴⁵Ca was studied using everted sacs of duodenum and ileum. Diabetic rats were compared with matched controls (diabetes studies); vitamin D-depleted animals were compared with depleted animals repleted with vitamin D 48 hrs prior to study (vitamin D studies). Duodenal calcium transport was reduced by half or less in experimental groups (diabetic or vitamin D-depleted rats) when compared with corresponding controls (control rats matched with diabetics or vitamin D-repleted animals matched with depleted animals):a) for serosal-to-mucosal (S/M) concentration ratios; andb) per unit weight of mucosa as net transport into serosal medium or net disappearance from mucosal medium. Based on transport into the serosal medium per unit of mucosa, ileal calcium transport was decreased in the vitamin D-depleted group compared with the vitamin D-repleted group in vitamin D studies. In contrast, in diabetes studies ileal calcium transport into the serosal medium was the same in diabetic and control animals. The finding that diabetes and vitamin D depletion have the same effects on duodenal calcium transport agrees witha) lack of specific, vitamin D-dependent duodenal calcium-binding protein in diabetes, andb) restoration of duodenal calcium transport in diabetics by either 1.25-dihydroxycholecalciferol or 1α-hydroxycholecalciferol. Depression of ileal calcium transport by vitamin D-depletion but not by diabetes shows the specificity of intestinal site with respect to mechanisms and control of calcium transport. Diabetes complements vitamin D depletion as a means for studying intestinal calcium transport. Supported in part by Research Grant 16841 from the National Institute of Arthritis, Metabolic and Digestive Diseases, National Institutes of Health.     

Vitamin D and intestinal calcium absorption

The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.     

Regulation by Calcium of In Vivo Synthesis of 1,25-Dihydroxycholecalciferol and 21,25-Dihydroxycholecalciferol

Tritiated 1,25-dihydroxycholecalciferol accumulates in several tissues, to an extent that varies with dietary calcium content, 12 hr after the administration of 325 pmoles of tritiated 25-hydroxycholecalciferol to rats. As the dietary and serum calcium concentrations increase, the amount of 1,25-dihydroxycholecalciferol is diminished and the concentration of 21,25-dihydroxycholecalciferol increases. This correlation is especially evident in rats given vitamin D(3). In vitamin D-deficient rats, the repression of 1,25-dihydroxycholecalciferol formation occurs with a diet containing 3% calcium and 20% lactose. The results suggest that the production of 1,25-dihydroxycholecalciferol, believed to be the metabolically active form of vitamin D in the intestine, is responsible for the adaptation of calcium absorption to low dietary concentrations of calcium.     

Inhibition of iron absorption by cholestyramine

Recent studies have demonstrated that cholestyramine, an anionic exchange resin, binds inorganic and hemoglobin ironin vitro and inhibits the absorption of these compounds from the rat small intestine. However, these studies did not answer the question: Will prolonged cholestyramine administration cause depletion of iron stores and development of iron deficiency anemia? Accordingly, rats were fed diets of standard rat chow or standard rat chow containing either 2 or 5% cholestyramine for 6 months. In the 5% cholestyramine group the hematocrit and hemoglobin values were significantly lower as compared to controls (37.5 vs 43.6%;P<0.001) and (11.4 vs 14.2 g%;P<0.001). Serum iron values in the 5% cholestyramine groups were significantly less than the control group (158±23 vs 205±15 µg%;P<0.001). In the control group nonheme iron in the liver, spleen and duodenum was 206±31, 1482±212 and 12.5±1.1 µg/g tissue, respectively. In the 2% cholestyramine-treated group, liver nonheme iron was reduced to 172±33 µg/g, spleen nonheme iron to 1266±447 µg/g and duodenal iron to 11.7±1.0 µg/g. In the 5% cholestyramine-treated group liver nonheme iron was significantly reduced to 143±35 µg/g, spleen nonheme iron to 1030±3.4 µg/g and duodenal nonheme iron to 10.2±1.4 µg/g respectively. In addition, a significant decrease in serum vitamin B₁₂ levels was noted in cholestyramine-fed rats compared to controls (256±83 vs 404±118 µµg/ml). These data indicate that prolonged administration of cholestyramine to rats results in significantly decreased serum iron, tissue nonheme iron stores and serum vitamin B₁₂ levels associated with decreased hematocrit and hemoglobin levels.     

TRPV6 is not required for 1α,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo

The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D₃ in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D₃ in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.