Inhibitory effect of αS1- and αS2-casein hydrolysates on angiotensin I-converting enzyme in human endothelial cells in vitro, rat aortic tissue ex vivo, and renovascular hypertensive rats in vivo.

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from alpha(S1)- and alpha(S2)-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood.

Effect of chronic renal failure on the expression and function of rat intestinal P-glycoprotein in drug excretion.

BACKGROUND: In chronic renal failure, the renal excretion of certain drugs is dramatically reduced. To determine whether other routes of drug elimination, such as secretion through the intestinal barrier by intestinal P-glycoprotein can be altered, we compared P-glycoprotein activity, P-glycoprotein protein content, and P-glycoprotein mRNA levels in intestine of control and chronic renal failure rats. METHODS: Chronic renal failure was surgically induced in rats by partial (7/8) nephrectomy. After 5 weeks, intestinal transport of rhodamine 123, a P-glycoprotein substrate, was carried out using an in vitro model of everted gut sacs. P-glycoprotein protein content was quantified by enzyme-linked immunosorbent assay and P-glycoprotein mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction. RESULTS: A decrease of intestinal rhodamine 123 transport was observed in chronic renal failure rats, pointing to an inhibition of P-glycoprotein activity. Transport was inhibited in both sham-operated rats and rats with chronic renal failure by verapamil and cyclosporin A, but relative inhibition vs baseline was less marked in chronic renal failure than in sham-operated rats. In contrast, no significant differences in levels of P-glycoprotein protein or mRNA were observed between the two groups. CONCLUSIONS: Intestinal secretion of rhodamine 123 is mainly mediated by P-glycoprotein. It was reduced in rats with chronic renal failure, reflecting reduced intestinal drug elimination via a decrease in P-glycoprotein transport activity rather than via protein underexpression.

Alpha-1-microglobulin: inhibitory effect on calcium oxalate crystallization in vitro and decreased urinary concentration in calcium oxalate stone formers.

In the past few years, alpha-1-microglobulin (alpha1m) has been copurified from human urine with bikunin, a potent inhibitor of calcium oxalate (CaOx) crystallization in vitro. In this study, we have purified alpha1m without bikunin contamination and investigated its possible role in CaOx crystallization by in vitro and in vivo studies. Alpha-1m was purified with an anti-alpha1m antibodies CNBr-activated sepharose column. Two molecular species of alpha1m of respectively 30 and 60 kDa were purified. For each protein, two blots of 30 and 60 kDa cross-reacted with anti-alpha1m antibodies, suggesting that these two forms were derived one from the other. Both protein species inhibited CaOx crystallization in a dose-dependent manner in two in vitro tests. In the first test, the presence of alpha1m of 30 kDa (8 microg/ml) in a medium containing 0. 76 mM CaCl(2) (with (45)Ca) and 0.76 mM Ox(NH(4))(2) inhibited CaOx crystallization by 38% as estimated by supernatant radioactivity after 1 h of agitation. In the second test, CaOx kinetics were examined for 3 to 10 min in a turbidimetric model at 620 nm. The presence of alpha1m of 30 kDa in a medium containing 4 mM CaCl(2) and 0.5 mM Na(2)Ox inhibited CaOx crystallization by 41.5%, as estimated by the slope modification of turbidimetric curve. Alpha-1m can be considered as another inhibitor of urinary CaOx crystal formation, as shown by the present in vitro studies. Using an ELISA assay, we found that urinary alpha1m concentration was significantly lower in 31 CaOx stone formers than in 18 healthy subjects (2.95 +/- 0.29 vs 5.34 +/- 1.08 mg/l respectively, P = 0.01). The decreased concentration of alpha1m in CaOx stone formers could be responsible in these patients, at least in part, for an increased risk of CaOx crystalluria.

Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers.

Two proteins of 17 and 24 kDa, respectively, which were immunologically related to bikunin, were purified from urine of healthy men, using in the last step a trypsin CNBr-sepharose affinity column. These proteins strongly inhibited calcium oxalate (CaOx) crystallization in two in vitro models. In the first model, the presence of 8 microg/ml protein in a medium containing 0.76 mM CaCl2 (with 45Ca) and 0.76 mM ammonium oxalate inhibited the crystallization process by 80%, as estimated by supernatant radioactivity after 60 min of incubation. A similar inhibition was observed in the second turbidimetric model, where the CaOx crystallization kinetics were followed for 10 min at 620 nm in a medium containing 4 mM CaCl2 and 0.5 mM Na2Ox. These proteins were used as standard protein for the development of an enzyme-linked immunosorbent assay (ELISA) in urine. Mean (+/-SEM) urinary bikunin concentration in 18 healthy subjects was 5.01 +/- 0.91 microg/ml. This was a concentration range of strong inhibitory activity in vitro. Bikunin values were nearly 50% lower (2.54 +/- 0.42 microg/ml, P=0.007) in 31 CaOx renal stone formers (having weddelite crystals in their first morning urine) than in the healthy volunteers. A correlation was found between urinary bikunin and alpha-1 microglobulin concentrations in the control group (y=0.73x + 1.09, r2=0.8) while no such correlation existed in the lithiasis group. In conclusion, bikunin exerts a strong inhibitory action of CaOx crystallization in vitro. Its involvement in urinary CaOx crystallization of stone formers is highly probable, based on the significant decrease in its urinary concentration in the majority of stone formers studied.

Role of transcellular pathway in ileal Ca2+ absorption: stimulation by low-Ca2+ diet.

The present study was performed to determine the respective involvement of the cellular and paracellular routes in ileal Ca2+ transport. Two groups of rats were either fed a normal Ca2+ diet (1. 0%) or a Ca2+-deficient diet (0.02%) for 14 days. Ileal Ca2+ absorption was determined using both an in situ method of continuous luminal perfusion and an in vitro method (Ussing chamber model). The low-Ca2+ diet stimulated net Ca2+ flux in the ileum twofold, associated with a twofold increase of the mucosal-to-serosal Ca2+ flux in both models. This effect was observed in the absence of concomitant changes in Na+ or water flux in the in situ model or mannitol flux in the in vitro model, excluding the participation of the paracellular pathway in Ca2+ transport. Thus only cellular Ca2+ flux was stimulated. These data suggest that the ileum plays a major role in the adaptation to low dietary Ca2+. Whereas under physiological conditions with usual Ca2+ intakes the transcellular pathway of Ca2+ transport is negligible, it becomes of major importance in the case of Ca2+ deficiency, at least under the present conditions of severe Ca2+ deprivation.

A stable animal model of diet-induced calcium oxalate crystalluria.

Twenty male Wistar rats, weighing 150 g, were placed in metabolic cages on a 30% sucrose diet for 7 days, before allocation to two groups: a control group (n = 5) and a lactose group (n = 15). They received respectively a 30% sucrose diet or a 30% lactose diet for 8 weeks, each containing 0.67% calcium and 0.38% phosphorus. After 4 (T1) and 8 (T2) weeks, the serum calcium (Ca) and citrate levels were significantly (P < 0.01) higher in rats fed the lactose diet. Serum alkaline phosphatase activity was increased in the lactose group (P < 0.01) at T1 and T2. The lactose-rich diet induced an increase in urinary Ca excretion at T1 and T2; citrate excretion was only enhanced at T2 (P < 0.001). No difference between the two groups was observed in urinary oxalate (Ox) excretion or creatinine clearance. Crystalluria analysis revealed a marked number (>300/mm3 at T1 and T2) of calcium oxalate dihydrate crystals (COD) in rats fed the lactose-rich diet, whereas no COD crystals were observed in sucrose-fed control rats at any time point. The formation of COD crystals in lactose-fed rats was related to an increase in calcium oxalate (CaOx) product (pCaOx), which was respectively 12.6 vs 3.9 at T1 and 10.5 vs 1.8 at T2, and an increase in CaOx ratio (Ca/Ox), which was 99.1 vs 7.5 and 67.5 vs 18.5 at T1 and T2, respectively. The high pCaOx and Ca/Ox ratios in the lactose group were due to hypercalciuria, in agreement with the number and the type of crystals. The present experimental model confirms that the ingestion of a 30% lactose diet increases urinary Ca excretion without changing urinary Ox excretion and shows for the first time that it induces a stable and marked crystalluria composed of COD. Such a non-nephrotoxic and stable model is of interest for the study of CaOx crystal formation secondary to hypercalciuria, and thus afterwards eventually for CaOx nephrolithiasis.

Stimulation by citric acid of calcium and phosphorus bioavailability in rats fed a calcium-rich diet.

The effect of dietary citric acid supplementation on calcium (Ca) and phosphorus (P) bioavailability was studied in rats fed 3 different diets containing 0.1, 0.5 or 1.0 g/100g Ca during 7 weeks. Citric acid supplementation increased intestinal Ca and P absorption and the Ca and P retention/intake ratio only in rats fed the 1% Ca diet. It also increased the P concentration of bone ashes in rats fed the 0.5% Ca diet (18.9 +/- 0.2 vs 17.5 +/- 0.5%) and the 1% Ca diet (20 +/- 0.1 vs. 19 +/- 0.3%), and the Ca bone ash concentration in rats fed the 1% Ca diet (36.7 +/- 0.4 vs. 35.7 +/- 0.5%). In rats fed the 1% Ca diet, plasma P concentration was decreased by citric acid supplementation (2.09 +/- 0.10 vs. 2.45 +/- 0.08 mmol/l) while urinary P excretion was increased (18.2 +/- 2.3 vs. 2.0 +/- 0.3 mmol/4 days), together with a decrease in plasma calcitriol concentration (54.4 +/- 2.6 vs. 79.6 +/- 2.5 ng/l), but no change of the circulating parathyroid hormone level. This study indicates that citric acid supplementation together with a Ca-rich diet allows to obtain an increased retention of Ca and P in bone. The prolonged administration of Ca citrate supplements may therefore help to increase bone mineral concentration.

[Stimulation of ileal transport of calcium by sorbitol in in situ perfused loop in rats]. / Stimulation du transport iléal du calcium par le sorbitol en anse perfusée in situ chez le rat.

OBJECTIVES: The aim of this study was to study the effect of sorbitol on sodium, water and calcium fluxes in rat ileum in situ perfused loop. METHODS: Net water, sodium and calcium fluxes, and one-way calcium fluxes were measured in situ in a perfused rat ileal loop in the presence of varying concentrations of sorbital. RESULTS: High concentrations of sorbitol in perfused ileal solution induced a decrease of sodium and water fluxes and a concomitant increase of lumen to mucosa calcium flux associated with an increase of net calcium flux, using a solution containing either 8.0 or 1.25 mM calcium. These effects were independent of absolute initial values of water and sodium fluxes. They were observed in the presence of 25 mM glucose, 10 mM theophyllin or after treatment of rats with dexamethasone. CONCLUSION: These effects of sorbitol on calcium flux are not compatible with a stimulation of paracellular pathway. By contrast, they can be explained by a stimulation of transcellular calcium pathway in ileum associated with the hyperpolarisation of the cells induced by the decrease of luminal sodium concentration necessary in the presence of sorbitol to maintain unchanged osmolarity of perfusate.

Mechanism of the enhancing effect of sorbitol on ileal Ca uptake in rat enterocytes.

The effect of sorbitol on Ca uptake by isolated ileal epithelial cells was investigated. Intestinal cells were isolated from rat ileum by mechanical vibration. 45Ca uptake was approximately 2 times higher in cells exposed to 200 mM sorbitol of D-alanine than in control cells. This enhancing effect of sorbitol on percentage Ca uptake decreased with increasing Ca concentrations in the incubation medium suggesting an effect on Ca entry velocity. The addition of 10 microM nifedipine or 200 microM verapamil to the incubation medium was devoid of any effect on Ca uptake in ileal cells, whereas 100 microM trifluoperazine or chlorpromazine abolished the stimulatory effect of sorbitol. Finally, the effect of sorbitol on isolated cells was independent of a measurable change of cellular ATP content. In conclusion, the stimulatory effect of sorbitol on ileal Ca uptake is probably exerted through mechanisms other than an increase in intracellular ATP concentration. Sorbitol may enhance enterocyte Ca transport via a direct interaction with calmodulin and/or the Ca pump. It may also exert its effect through an inhibition of the basolateral Na Ca exchanger.

Stimulation of ileal calcium absorption by sorbitol, L-xylose, or creatine via a decrease in luminal sodium concentration: relation with concomitant changes in enterocyte energy metabolism.

Ligated ileal loops, 30 cm in length, of 4-month-old male Wistar rats were instilled with 3 ml of a 10 mM CaCl2 solution (added with 0.25 muCi 45Ca) in the absence (control) or presence of 100mM sorbitol, L-xylose, or creatine. Ileal calcium (Ca) transport, measured by plasma 45Ca appearance, was found to be similar 30 minutes after fluid instillation in all four instances. However, thereafter, 45Ca appearance in plasma did not increase further in control animals whereas it increased twice as much during the subsequent 30 minutes in the presence of sorbitol, L-xylose, or creatine. However, when loops of similar length were instilled with only 1.0 ml of such solutions, the sorbitol effect was already observed during the first 30 minutes. The stimulation of ileal Ca absorption induced by the presence of sorbitol appeared to be due to a cellular effect, associated with a decreased flux across the paracellular pathway, as indicated by 3H-mannitol absorption. The presence of sorbitol in instilled ileal solution induced a significant decrease in luminal Na, K, bicarbonate, and Cl concentrations at each time point studied (30, 60, 120, or 240 minutes after instillation). Thirty minutes after instillation, no difference in soluble Ca concentration was observed between control and experimental rats. After 60 minutes, Ca concentration was dramatically decreased in control rats but it remained nearly constant in experimental animals. Thus, the presence of substances enhancing ileal Ca transport favored the maintenance of soluble Ca in ileal solution during longer time periods than their absence. In the ileal enterocyte, these substances induced a twofold increase of ATP content compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Different forms of alkaline phosphatase in adult rat femur. Effect of a vitamin D3-deficient diet and of a sorbitol-enriched diet.

In the femoral extremities of the adult rat containing the metaphysis, the epiphyseal cartilage, and the epiphysis, four alkaline phosphatase (AP) forms were distinguished on polyacrylamide gel electrophoresis. Two soluble forms were present in the 160,000 g supernatant: one of Mr 165 kDa and another of Mr 110-115 kDa, which exhibited a strong catalytical activity. Moreover, from the pellet, three membrane-bound forms of Mr 130, 110-115, and 100 kDa could be extacted with sodium deoxycholate. When denaturated AP was visualized by postelectrophoretic autoradiography of the phosphorylated intermediates, subunits always appeared as three monomers of Mr 75-80, 60-70, and 50-60 kDa. As four native forms but only three types of subunits were found to be present in the femur, it seems that, apart from homodimers, some heterodimers could also occur. Three types of diets were administered to three groups of rats for 5 weeks. Two are known to disturb bone mineralization: (1) a vitamin D3-deficient diet, and (2) the same as (1) but enriched with 12% sorbitol. The third was a normal diet containing vitamin D3. Concerning the effects on AP of dietary sorbitol and the vitamin D3-deficient diet, it was found that rats receiving the diet supplemented with sorbitol showed a substantial rise in the activity of the Mr 165 kDa form with the concomitant appearance of a new monomer of Mr 100 kDa. In contrast, rats fed the vitamin D3-deficient diet always displayed an increase in enzyme activity, principally of the Mr 100 and 110 kDa forms.(ABSTRACT TRUNCATED AT 250 WORDS)

[Metabolic effects of phosphate receptors stimulating the ileal transport of calcium]. / Effets métaboliques d'accepteurs de phosphate stimulant le transport iléal du calcium.

Several compounds, either metabolizable such as sorbitol and creatine or unmetabolizable such as L-xylose or ethyl-ethanolamine, increase ileal calcium transport, similarly to the well documented effect of lactose. These compounds increase the duration time but not the rate of calcium transport. They have no specific effect on intestinal calcium absorption but they prolong the presence of soluble calcium in the ileal loop, thus allowing the calcium absorption. This stimulating effect of these compounds on mineral absorption is associated with an increase of ileal mucosal ATP content.

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.

Modulation of ileal calcium transport by phosphate-exchanging compounds.

Calcium transport in the ileal-ligated loop was studied in the adult rat in the presence of either phosphate alone or phosphate-binding compounds, namely either hydroxylated or aminated substances. Sorbitol or creatine (50 mM) added to a 10-mM CaCl2 solution, which was instilled into ileal loop, markedly enhanced calcium transport, as determined by 45Ca radioactivity appearing in plasma and from 45Ca radioactivity disappearing from the loop. The presence of both compounds maintained Ca soluble in an instilled solution at a constant concentration, whereas with a control solution the Ca concentration progressively decreased towards zero after an incubation period of 60 min. Phosphate, which was either simultaneously added with sorbitol or creatine or which was present as sorbitol or creatine phosphate, led to an equally marked decrease in calcium transport. Calcium transfer was even more reduced when phosphate alone was present with calcium in the ileal loop, in the absence of sorbitol. Similar to the above phosphate-binding compounds, adenosine and its constitutive component, ribose, increased calcium transfer, whereas adenine, the other constitutive component of adenosine, was ineffective. Guanosine was twice more active than adenosine in stimulating ileal calcium transport. Interestingly, the structure of guanosine allows the binding of two phosphates, with one binding site being on the ribose and the other on the guanine base moiety. Thus guanosine is capable of binding a greater amount of phosphate than the two other aminated compounds examined, namely adenosine and alanine, when transphosphorylation from ATP is studied with intestinal microvilli preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal transfer of manganese: resemblance to and competition with calcium.

The effect of calcium, phosphate and the sugars lactose and sorbitol on the intestinal absorption of manganese were studied in adult male rats. Gastric gavage showed that lactose (100 mM or 200 mM) increased the hepatic retention of 54Mn, while phosphate decreased it. In situ ileal loop studies indicated that Mn absorption was normally complete in 30 min. Sorbitol had no effect on uptake during this period, but extended Mn absorption from 30 min to 120 min. Low concentrations of Mn (10 microM) did not alter the enhancing effect of lactose on calcium transport (10 mM), but the enhancing effect of lactose on Mn transport was blocked by this high calcium concentration. Intestinal alkaline phosphatase activity was rapidly stimulated by Mn. These similarities plus the competition between cations, especially calcium, suggest that a common mechanism exists in their intestinal transport.

Effect of some alkaline phosphatase inhibitors on intestinal calcium transfer.

1. There is a good correlation between the capacity of sugars to stimulate calcium transfer and their capacity to be phosphorylated by the intestinal alkaline phosphate with a part of the phosphate liberated from an ester phosphate. 2. On the sugar dependent and sugar independent calcium transfer, inhibitors of this enzyme act differently. 3. Phosphate, a competitive inhibitor suppresses both transfers. 4. Only the dependent sugar transfer was suppressed with phloridzin acting competitively at the sugar site, or with EDTA which could react close to the active site. 5. L-phenylalanine and phenobarbital, not competitive inhibitors does not act on either type of calcium transfer, the sugar dependent or the sugar independent.

Intestinal calcium transfer and alkaline phosphatase activity in relation with vitamin D and glucide diet.

For four weeks after weaning, rats were fed either on a diet without any calcium utilization factors (-D) or on the same diet with cholecalciferol (+D) or sorbitol (S). In the -D group, blood calcium levels decreased whilst alkaline phosphatase activities in blood and bone were increased. For +D and S groups, these parameters were normal. Using everted or in situ ligatured loops, calcium transfer from a CaCl2 + 45Ca solution was measured in the duodenum, the jejunum and in the ileum. Alkaline phosphatase activity from these regions was also measured. For the three diets and for all regions of the intestine, there was a good correlation between calcium transfer and phosphatase activity. These values were higher in the duodenum than in the ileum or jejunum, and also higher in the ileum in the +D group than in the -D and S groups although this was not significant. These low levels in the S group which were, sometimes, even lower than those seen in the -D group contrasted with blood and bone levels of alkaline phosphatase, which were normal for the S and +D groups. There was also a discrepancy between the low values found for both phosphatase activity and calcium transfer in rats S in the experiments where the calcium transfer assay was carried out in calcium solution and those found in experiments were both calcium and carbohydrate were present. In the latter, enhanced levels of intestinal phosphatase activity were observed, as well as a marked, albeit delayed, increase in intestinal calcium transfer. Onset latency and rapid offset are reminiscent of induction of bacterial enzymes by carbohydrates.

Alkaline phosphatase from adult rat femur.

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.

Changes of femoral alkaline phosphatase activity in adult rats treated by sorbitol enriched or vitamin D3 deficient diet.

The effect of vitamin D3-deficiency and dietary sorbitol on serum calcium level, the activity and alkaline phosphatase (AP) pattern in femoral epiphysis were studied. Rats fed a diet supplemented with sorbitol or vitamin D3 showed the same serum calcium concentration and AP activity in serum and femur. Rats fed a vitamin D3-deficient diet displayed decreased serum calcium concentration and increased AP activity both in serum and femur. Four forms of AP were isolated from the femur of these rat groups: of Mr 100,000, 110,000, 130,000 and 165,000. Rats receiving the diet supplemented with sorbitol showed a marked rise in the activity of the Mr 165,000 form, and appearance of a new monomer of 100,000, never formed in two remaining groups.

Differential effect of ethanol on formation of phosphorylated intermediates of the jejunal brush border alkaline phosphatase Mr 90,000 and 65,000 subunits.

The influence of ethanol on formation of phosphorylated intermediates of different forms of alkaline phosphates in brush border of adult rat was studied. Ethanol enhanced phosphorylation of the Mr 65,000 subunit of jejunal enzyme but had no effect on the Mr 90,000 subunit. Commercial highly purified alkaline phosphate in the presence of ethanol also displayed a two times higher phosphorylation ability.