DIETAS BAJAS EN CALCIO EN EL HOMBRE Y EN ANIMALES DE EXPERIMENTACIÓN; MODELOS CLÁSICOS PARA ENTENDER LA HOMEÓSTASIS CÁLCICA Y EL SISTEMA ENDOCRINOLÓGICO DE LA VITAMINA D

El objetivo de este artículo es revisar la contribución aportada por el uso de dietas bajas en calcio, en el hombre y en animales de experimentación, para esclarecer la homeóstasis del calcio y del papel de la vitamina D. Se hace un énfasis especial en el efecto de estas dietas pobres en calcio sobre el metabolismo de la vitamina D, la síntesis y la secreción paratiroidea, el número de células paratiroideas y las propiedades químicas y físicas de las membranas plasmáticas de las células intestinales. También se discuten los datos referentes a la interacción entre el genotipo del receptor de la vitamina D y de la ingesta de Ca sobre la absorción intestinal de este mineral.

The aim of this article is to review the contribution performed by the use of low Ca diets either in humans or experimental animals to the knowledge of Ca homeostasis and the role of vitamin D. Special emphasis will be focused on the effect of these mineral restricted diets on vitamin D metabolism, parathyroid hormone (PTH) synthesis and secretion, number of parathyroid cells and chemical and physical properties of the intestinal plasma membranes. A brief analysis of the data regarding to the interaction between the vitamin D receptor (VDR) genotype and Ca intakes on intestinal Ca absorption will be also discussed.

The main role of the vitamin D related hormone is the regulation of intestinal Ca²⁺ absorption and Ca²⁺ homeostasis¹. Being the diet the only source of Ca²⁺, adaptive mechanisms have evolved to control the amount of dietary calcium that is absorbed. When humans or experimental animals have low Ca intakes, the efficiency of intestinal Ca absorption increases^2,3. This mechanism of adaptation depends on the vitamin D status, mainly of the rate of synthesis of 1,25 (OH)₂D₃^4-6. Increment of calbindin D_9k in mammals and calbindin D_28k in birds (cytosolic proteins presumably involved in the transcellular movement of Ca²⁺) as well as higher levels of their respective mRNA are induced by dietary Ca²⁺ deficien-

cy^7-9. Low Ca diets not only affect vitamin D metabolism, but also parathyroid hormone (PTH) metabolism and parathyroid cells¹⁰. In addition, molecular and biophysical changes occur in the plasma membrane of intestinal epithelial cells which may be involved in the global process of intestinal Ca absorption increment^11,12. Recent studies indicate that intestinal Ca absorption is also influenced by interactions between vitamin D receptor (VDR) genotype and environmental factors such as dietary calcium and vitamin D¹³. A more detailed analysis of these issues will be explained in the following sections.

EFFECT OF DIETARY CALCIUM RESTRICTION ON VITAMIN D METABOLISM

Cholecalciferol undergoes metabolic conversion before it exerts its biological effects. It is hydroxylated in two steps: the first hydroxylation occurs at the level of C-25 in the liver, and the second hydroxylation, which is produced in the kidney, is on the C-1 leading to the production of 1,25 (OH)₂D₃ (calcitriol), the hormone derived from vitamin D. Another hydroxylation takes place in kidney and in other tissues under certain physiological conditions, producing the metabolite 24,25(OH)₂D₃. More than 40 metabolites are synthesized from vitamin D, but, 1,25(OH)₂D₃ is the molecule responsible for the main actions of vitamin D¹⁴.

Feeding a low Ca diet results in significant stimulation of 1.25-dihydroxyvitamin D₃ production in young rats, but not in aged rats¹⁵. During the early stages of calcium deficiency, serum levels of PTH and 1,25 (OH)₂D₃ increase in parallel and, after 3 weeks of Ca depletion, levels of calcitriol decline somewhat, but remain fourfold higher than those measured initially for about 6 weeks¹⁰. The increase in serum levels of 1,25 (OH)₂D₃ by a low Ca diet has been demonstrated in humans¹⁶, chicks³, ewes¹⁷ and rats⁴. Schedl et al¹⁸ did not find increased levels of calcitriol in hamsters fed a low Ca diet although the ileum calcium absorption was increased.

It is well established that PTH, secreted in response to hypocalcemia, causes a stimulatory effect of 1,25 (OH)₂D₃ synthesis¹⁹. However, when animals are given a low Ca and high vitamin D diet, the ability of PTH to increase kidney 1-hydroxylase is blunted and the metabolic clearance of 1,25 (OH)₂D₃ is enhanced²⁰. Bell²¹ suggests that the renal synthesis of 1,25 (OH)₂D₃ is not only modulated by PTH but also by the calcium receptor that has been recently described in kidney.

Mortensen et al²² have found that the toxicity of 1- * (OH) D₃, a synthetic analogue of 1,25 (OH)₂D₃, was reduced in rats fed a semi-synthetic low-Ca diet compared with rats fed standard diet. The authors speculate that the use of diets low in Ca could allow the administration of higher doses of vitamin D derivatives or analogues without causing hypercalcemia.

The number of VDR is also influenced by dietary calcium restriction²³. In contrast to the exogenous administration of 1,25 (OH)₂D₃, when high plasma levels of 1,25 (OH)₂ D₃ are achieved by endogenous production of 1,25 (OH)₂D₃ in response to chronic restriction of dietary calcium, intestinal VDR is not up-regulated and renal VDR content is down-regulated which indicates that some factor other than 1,25 (OH)₂D₃ plays a role in regulating VDR content of tissues²⁴.

The effect of low Ca diet on other metabolites derived from vitamin D is unclear. Fox et al²⁵ did not show increase in the 1,25 (OH)₂D₃ catabolism of rats fed a low Ca diet, but the metabolic renal clearance of 1,25 (OH)₂D₃ was accelerated by dietary calcium restriction. On the contrary, Goff et al²⁶ detected that dietary Ca restriction increases intestinal 24-hydroxylase activity 6 to 20-fold above that of rats fed a Ca replete diet.

INFLUENCE OF DIETARY CALCIUM DEPRIVATION ON PARATHYROID CELLS AND PTH METABOLISM

It is well documented that PTH secretion is regulated by calcium, being the secretion stimulated by low and inhibited by high calcium levels²⁷. A sigmoidal relationship between PTH secretion and serum calcium has been confirmed in humans and experimental animals^28,29. Brown³⁰ defined the set-point of this relationship, which is the calcium concentration producing half of the maximal inhibition of secretion. This parameter is useful in the analysis of PTH from patients with secondary hyperparathyroidism due to renal failure and in other conditions.

The sensitivity of parathyroid glands is remarkable; small changes in serum calcium produce large changes in PTH secretion³¹. A parathyroid calcium sensor has been recently characterized and cloned³². This protein would mediate the physiological responses of the parathyroid cell to calcium. A variety of mutations inactivating the Ca sensor gene have been demonstrated in patients with familial hypocalciuric hypercalcemia³¹.

Low Ca diets affect parathyroid cell proliferation, regulation of PTH gene expression and PTH secretion. Naveh-Many et al³³ have found that a Ca restricted diet leads to increased levels of PTH mRNA and a 10-fold incremented in parathyroid cell proliferation.

Sela-Brown et al³⁴ pointed out that the interrelationship of the effect of calcium and 1,25 (OH)₂D₃ on the PTH gene and parathyroid cells is paradoxical. For instance, a dietary calcium deficiency results in low serum calcium leading to a marked, increment in serum 1,25 (OH)₂D₃, which would be expected to decrease PTH gene transcription. This dietary hypocalcemia is instead associated with an increase in PTH mRNA and serum PTH levels³¹. Preliminary data indicate that the effect of Ca²⁺ on PTH mRNA levels in vivo occurs with no detectable changes in PTH gene transcription rate³⁵. The results suggest that the effect of calcium in vivo involves post-transcriptional mechanisms. Studies in vitro show that the 3'-untranslaated region of the PTH mRNA binds cytosolic proteins which may be involved in the post-transcriptional increase in PTH gene expression induced by hypocalcemia. These parathyroid proteins added in vitro to labeled PTH transcripts affect the transcript half-life. Hypocalcemic parathyroid proteins kept the transcript intact for a much longer period than those from control animals indicating that parathyroid proteins from hypocalcemic rats protect the PTH mRNA from degradation³¹. Recently, it has been shown that calreticulin, a protein that binds to the conserved sequence KXGFFKR of steroid hormone receptors altering transcription of steroid responsive genes, is highly increased in the nuclear fraction of parathyroid gland and not in other tissues from hypocalcemic rats³⁴. The data suggest that calreticulin may prevent the transcriptional effect of 1,25 (OH)₂D₃ on the PTH promoter. The effect of calreticulin might explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to calcitriol treatment.

EFFECT OF DIETARY CALCIUM DEFICIENCY ON COMPOSITION OF INTESTINAL PLASMA MEMBRANES AND CALCIUM TRANSPORT

Lipid and protein composition of plasma membranes from enterocytes have been demonstrated to be altered by Ca²⁺ restriction in the diet. Krawitt et al³⁶ found that Ca²⁺ transport, measured by an in vitro gut sac technique, was increased in duodenum, jejunum and terminal ileum of rats fed a calcium-deficient diet for 7 days. They also observed that alkaline phosphatase activity was increased in the duodenum. They suggested that if alkaline phosphatase plays a role in this adaptation, it would be limited to the duodenal segment and might involve a process independent of active transport. Recently, in our laboratory, we have demonstrated that a low Ca diet increases the activity of this enzyme either in mature or in undifferentiated intestinal absorptive cells from chick duodena. The Ca²⁺ uptake by enterocytes was also enhanced in both cell types³⁷.

The reactivity and availability of sulfhydryl groups from proteins of intestinal brush border membranes in chicks adapted to a low Ca diet were analyzed. By the Ellman

reaction, a threefold increment in HS^­ group content was noted. By using DACM (N-7-dimethylamino-4-methylcoumarin-3-yl-maleimide), a fluorescent probe for HS^­ groups, an adduct was formed which developed fluorescence. Fluorescence intensity developed more rapidly when the brush border membranes were from the low calcium than when from the control group. The reactivity of the membrane from the low Ca group was greater in the presence of detergent, which presumably exposed buried sulfhydryl groups. Both the maximum fluorescence intensity and the pseudo-first-order rate constant of the DACM reaction with brush border membranes (BBM) were higher in the Ca restricted group than in the control one¹¹. Previous results had shown similar changes in BBM sulfhydryl groups when 1,25 (OH)₂D₃ was given to vitamin D-deficient chicks³⁸. Although the functional significance of the increment in HS^­ groups promoted by the low Ca diet remains unknown, it is reasonable to think that the sulfhydryl status of the BBM proteins could be involved in the vitamin D-dependent intestinal absorption of calcium.

Low Ca diet in experimental animals also causes changes in the two main proteins involved in the Ca²⁺ extrusion at the intestinal basolateral membranes (BLM): plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger. Regarding to the former protein, it has been demonstrated by using a monoclonal antibody against the human erythrocyte Ca²⁺ pump that cross reacts with the chick intestinal Ca²⁺ pump epitope, that either low Ca or low P diet incrases the number of intestinal plasma membrane Ca²⁺ pump units as occurs after vitamin D administration to D-deficient chicks³⁹. This appeared physiologically reasonable because of the stimulating effect of vitamin D and low Ca or low P diets on the intestinal Ca²⁺ absorption. Later on, it was demonstrated that Ca²⁺ pump mRNA concentration is increased by 1,25(OH)₂D₃ due to an enhancement of the transcription rate of the Ca²⁺ pump gene^40,41. Recent data from our laboratory show that the activity of the intestinal Na⁺/Ca²⁺ exchanger is also enhanced by feeding chicks a low Ca diet for 10 days. The activity of this exchanger was modified in enterocytes from the apex but not in cells from the crypt of the intestinal villus⁴². Besides, it has been shown that lipid composition and fluidity of intestinal BLM are also altered by dietary calcium deficiency. Minor changes in the fatty acid content of the BLM were produced by the low Ca diet, such as a decrease in palmitic acid content and increase in the 22:5n3 and 22:6n3 fatty acids. However, by measurements of

steady-state fluorescence anisotropy, it was demonstrated that the lipid fluidity of diphenylhexatriene-labeled intestinal BLM was highly increased by the Ca²⁺ restriction in comparison to that of the control group¹². Thus, it appears that the Ca²⁺ exit through the BLM from the enterocytes in the animals adapted to a low Ca diet is greater than that from the control group because of the increment in the activity of the Ca²⁺ pump and Na⁺/Ca²⁺ exchanger and changes in lipid composition and fluidity of BLM which could affect the microdomains of ion transporters and, hence, increase their activities.

INTERRELATIONSHIP BETWEEN CALCIUM ABSORPTION, LOW CALCIUM INTAKE AND VITAMIN D RECEPTOR GENOTYPE

Genetic polymorphisms have been identified in the human VDR gene. In the past 5 years, a great body of information appeared in relation to the genetic polymorphisms defined within the intron between exons 7 and 8 as well as within the 3'untranslated region of exon 9⁴³. These polymorphisms seem to be correlated with bone mineral density (BMD) in several human populations and it was hypothesized that the determination of VDR genotypes could be useful as a predictor of development of osteoporosis⁴⁴. For instance, by using the restriction enzyme Bsm I on a DNA fragment amplified by the polymerase chain reaction wich includes the polymorphic Bsm I site in intron 7 of the gene, it is possible to determine 3 different genotypes: BB, Bb and bb. Genotype BB represents homozygous absence of the Bsm I restriction site, bb the homozygous presence of that site and Bb the heterozygous genotype. It has been found that women with the bb genotype had the highest BMD⁴⁵. The genotypes could be ranked as BB < Bb < bb in relation to BMD. However, negative and positive studies make this issue very controversial at present.

The relationship between VDR genotypes and physiological parameters appears to be dependent on environmental factors like calcium and vitamin D intake and gene-environment interactions. It has been found that the calcium absorption of the BBAA haplotype (defined by restriction endonucleases Bsm I and Apa I) was lower than that of the homozygous haplotype (bbaa). Krall et al⁴⁶ demonstrated influence of years elapsed since menopause and Ca intake on VDR alleles and rates of bone loss. They observed that the low Ca intake in healthy postmenopausal women increased the adverse association of the BB genotype with rapid femoral neck bone loss. The failure of the BB genotype to maintain calcium balance during Ca deficiency may result from an altered 1,25 (OH)₂D₃ receptor status and inability to produce the positive adaptation of increasing the intestinal Ca absorption. Ferrari et al⁴⁷ studied the relationship between VDR gene polymorphism and BMD in females from prepuberty to menopause and prospectively investigated the interaction of VDR genotypes with dietary Ca and BMD during childhood. In bb girls the BMD gain appeared to be higher than among the other genotypes when dietary Ca intake was low. BMD was significantly associated with VDR gene polymorphisms only before puberty. By increasing dietary Ca intake, BMD accrual was increased in Bb and BB prepubertal girls whereas bb subjects had the highest spontaneous BMD accrual and were not affected by Ca supplements. In summary, most of the recent studies reveal a consistent role of VDR alleles on intestinal Ca absorption and interaction with Ca intake even though the underlying molecular mechanisms remain unknown.