Biomedical subjects

T Thuren

Publications and source records attributed to T Thuren.

At least 19 recordsLinked to original sources

PubMed2000

Hepatic lipase and HDL metabolism.

Hepatic lipase is a lipolytic enzyme that has been suggested to have a role in HDL metabolism. Evidence suggests that HDL-cholesterol level is at least partly regulated by hepatic lipase level. Recent studies have shown that hepatic lipase not only hydrolyzes triglyceride and phospholipid in HDL, but also stimulates HDL cholesterol ester uptake by hepatocytes. Therefore, hepatic lipase, together with lipid transfer proteins, determines both HDL-cholesterol level and its function in reverse cholesterol transport. These conclusions are based on observations from in-vitro model substrate studies, cell culture studies, transgenic animal studies, and clinical studies. At present time, it is not known whether hepatic lipase action increases or decreases risk of developing atherosclerosis.

Animals↗

PubMed1995

Biosynthetic conversion of phosphatidylglycerol to sn-1:sn-1' bis(monoacylglycerol) phosphate in a macrophage-like cell line.

Bis(monoacylglycerol) phosphate has a unique stereoconfiguration of sn-glycero-1-phospho-1'-sn-glycerol and is synthesized from exogenous phosphatidylglycerol by macrophages. Previous work by our laboratory showed that the macrophage-like cell line RAW 264.7 synthesizes sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate. Here we describe studies using RAW 264.7 cells that examine the biosynthetic pathway by which bis(monoacylglycerol) phosphate is formed. Experiments were conducted using precursors that were specifically radiolabeled on the glycerol backbone in order to examine the stereoconfiguration of the intermediates and products formed in intact RAW 264.7 cells. The results of our studies indicate that a complex series of reactions are involved in the synthesis of bis(monoacylglycerol) phosphate. In this proposed pathway phosphatidylglycerol is hydrolyzed to form 1-acyllysophosphatidylglycerol which is then acylated on the headgroup glycerol to form the sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate. The sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate is then thought to undergo a stereoconversion that proceeds via the required removal of the acyl group at the sn-1 position. The resulting sn-glycero-1-phospho-1'-sn-glycerol enantiomer of lysophosphatidylglycerol with the acyl moiety on the original headgroup glycerol is then acylated to form sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate.

Animals↗

PubMed1994

Lipoprotein lipase domain function.

Human lipoprotein lipase (LPL) monomer consists of two domains, a larger NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a major determinant of heparin binding. Analyses of NH2-terminal domain function were performed after site-directed mutagenesis of the putative active-site serine residue, while COOH-terminal domain function was assessed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cysteine, or glycine were transiently expressed in COS-7 cells. Mutant proteins were synthesized and secreted at levels comparable to native LPL; however, none of the mutants retained enzymatic activity. The mutant with alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable to native LPL. These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of the smaller COOH-terminal domain, LPL lipolytic and esterolytic activities as well as heparin binding properties were determined after reaction with a monoclonal antibody specific for this domain. Lipolytic activity was inhibited by the monoclonal antibody, whereas esterolytic activity was only marginally affected, indicating that the LPL COOH-terminal domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, suggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) binding of antibody did not cause dimer dissociation. A model is proposed for the two LPL domains fulfilling different roles in the lipolytic process.

Animals↗

PubMed1993

Regulation of rat hepatic lipase by the composition of monomolecular films of lipid.

The regulation of hepatic lipase (HL) by the lipid composition of monomolecular substrate films was examined using a monolayer technique at constant surface pressure. HL-catalyzed hydrolysis of triacylglycerol, a poor substrate for HL in pure monomolecular films, was activated by diradylglycerol and its phosphorylated derivatives in mixed films containing 10 mol % triacylglycerol. When triacylglycerol was progressively diluted with dialkylglycerol, triacylglycerol hydrolysis by HL was maximal between 90 and 98 mol % dialkylglycerol. The best activators, dialkylphosphatidic acid and dialkylphosphatidylethanolamine, increased triacylglycerol hydrolysis 13-14-fold, and the enhancement of HL-catalyzed triacylglycerol hydrolysis by the activator lipids was inversely related to the average mean molecular area of the mixed films. The hydrolysis of 5 mol % triacylglycerol in mixed films that also contained phosphatidylcholine and 0-20 mol % cholesterol was inhibited approximately 80% when the concentration of cholesterol was 10-13 mol %. Interestingly, between 15 and 17 mol % cholesterol the hydrolysis rate was restored to about 50% of the uninhibited rate, but at 20 mol % cholesterol this value decreased back to 80% inhibition of hydrolysis. The hydrolysis of phosphatidylethanolamine in mixed films with 0-20 mol % cholesterol decreased approximately 30% in films containing 10-12 mol % cholesterol. However, at 15 mol % cholesterol the hydrolysis rate was restored to the same level observed for a pure phosphatidylethanolamine film. This enhancement of HL activity occurred at about the same cholesterol concentration as the restoration of triacylglycerol hydrolysis observed for the triacylglycerol/phosphatidylcholine/cholesterol films.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Action of lysosomal phospholipase A1 on bis(monoacylglycerol)phosphate.

Bis(monoacylglycerol)phosphate (BMP) in macrophages is known to rapidly turn over its acyl moiety(s) located at primary positions of the glycerols, yet the glycerols and phosphate remain stable within the BMP molecule. Here we examine whether the phospholipase A1 isolated from rat-liver lysosomes is capable of deacylating BMP. By comparison with the precursor of BMP, phosphatidylglycerol, BMP is a very poor substrate for the phospholipase A1. We conclude, therefore, that a direct deacylation of the acyl groups at the primary alcohol level of the glycerol probably does not occur, but postulate that transacylations may occur to account for the removal of the acyl moiety.

Animals↗

PubMed1992

Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins.

We reported earlier that hepatic lipase (HL)-catalyzed hydrolysis of phospholipid monolayers is activated by apolipoprotein (apo) E [Thuren et al. (1991b) J. Biol. Chem. 266, 4853-4861]. On the basis of these studies, it was postulated that apoE-rich high-density lipoproteins (HDL) were preferred substrates for HL. In the present study, we tested this hypothesis, as well as further characterizing the activation of HL hydrolysis of phospholipid by apoE. The apoE-rich HDL, referred to as HDL-I, were isolated by heparin-Sepharose chromatography, and the phospholipid hydrolysis by HL was compared to an apoE-poor HDL, designated HDL-II. The hydrolysis of HDL-I phosphatidylcholine was approximately 3-fold higher than HDL-II, supporting the hypothesis that HL preferably hydrolyzes the phospholipids in apoE-rich HDL. In order to gain additional insight into the nature of the activation, we used phospholipid monolayers as model systems. Comparison of the ability of the two thrombolytic fragments of apoE (22 kDa, residues 1-191; 12 kDa, residues 192-299) revealed that only the 12-kDa fragment was capable of activating the hydrolysis of phospholipid by HL (1.75-fold). However, activation was less than with the intact protein (2.8-fold for apoE3), suggesting that the intact protein was required for full activation. The fact that the 12-kDa fragment, which represents a major lipid region of the protein, did activate HL suggests that activation occurs at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1991

Activation of hepatic lipase catalyzed phosphatidylcholine hydrolysis by apolipoprotein E.

The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.

Animals↗

PubMed1991

Glycerol reorientation during the conversion of phosphatidylglycerol to bis(monoacylglycerol)phosphate in macrophage-like RAW 264.7 cells.

Bis(monoacylglycero)phosphate (BMP) has the unique stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol] (Brotherus, J., Renkonen, O., Herrmann, J., and Fischer, W. (1974) Chem. Phys. Lipids 13, 178-182) which differs from other known mammalian phospholipids that have the sn-glycero-3-phosphoryl configuration. This stereochemistry may contribute to its physiologic function. Here we describe studies using the macrophage-like cell line RAW 264.7 designed to determined how this unique stereoconfiguration occurs. These studies show that the stereoconfiguration of BMP produced from exogenous phosphatidylglycerol (PG) by RAW 264.7 cells has the expected stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol]. Experiments using diacyl-sn-[2-3H]glycero-3-phosphoryl-sn-1'-[2-3H]glycerol demonstrate that this unique stereoconfiguration is not produced due to an oxidation/reduction mechanism involving the sn-2-glycerol carbon. When dioleoyl-sn-[1-14C]glycero-3-phosphoryl-rac-glycerol was converted to 14C-labeled BMP, the 14C label was found esterified to the phosphate moiety. These results suggest that a stereospecific enzyme is capable of reorienting the radiolabeled glycerol backbone of this PG substrate, effectively changing the stereochemistry of the lipid. We also show that this enzyme is stereoselective with regard to the base glycerol moiety of the substrate PG used. Finally, we propose a new pathway for the synthesis of BMP from PG.

Animals↗

PubMed1991

Hepatic lipase hydrolysis of lipid monolayers. Regulation by apolipoproteins.

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.

Animals↗

PubMed1991

Hydrolysis of neutral lipid substrates by rat hepatic lipase.

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Vmax values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The Vmax values obtained were 144 mumol/min/mg for 1-oleoyl-sn-glycerol, 163 mumol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 mumol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 mumol/min/mg for PA, 50 mumol/min/mg for PE and 4 mumol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at the sn-1 and sn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereoselective with respect to 1,3-diacyl-sn-glycerol substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

A continuous fluorometric assay for phospholipase C from Clostridium perfringens.

A fluorescent assay for Clostridium perfringens phospholipase C is described using 1-palmitoyl-2-[6(pyren-1-yl)hexanoyl]-sn-glycero-3- phospho-N-(trinitrophenyl)aminoethanol (PPHTE) as the substrate. This method is based on the decrease of the quenching of pyrene monomer fluorescence when phospholipase C hydrolyzes PPHTE into pyrenediglyceride and phospho(trinitrophenyl)-aminoethanol. The hydrolysis of egg lecithin/PPHTE (25:1 molar ratio) substrate by C. perfringens phospholipase C was linear with time for at least 2 min. Optimal conditions for the hydrolysis by phospholipase C were 50 mM Tris-HCl pH 7.0-30 mM CaCl2/63 microM egg lecithin and 2.5 microM PPHTE. The Km and Vmax values for the hydrolysis of egg lecithin/PPHTE vesicles were 28 microM and 280 pmol min-1, respectively. The detection limit of the assay was 40 microU of C. perfringens phospholipase C. When diglyceride was included into egg lecithin/PPHTE vesicles up to 30 mol% the reaction velocity increased 13-fold. Higher molar proportions of diglyceride were inhibitory. When the hydrolysis of mixtures of different naturally occurring phospholipids and PPHTE was studied egg lecithin was found to be the best substrate. When dipalmitoylphospholipids with different polar head groups were used the reaction velocity decreased in the order egg lecithin greater than or equal to dipalmitoylphosphatidylserine greater than dipalmitoylphosphatidic acid greater than dipalmitoylphosphatidylcholine greater than dipalmitoylphosphatidylglycerol.

Calcium Chloride↗

PubMed1990

Hydrolysis of lipid mixtures by rat hepatic lipase.

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Increased serum phospholipase A2 activity in schizophrenia: a replication study.

Phospholipase A2 (PLA2) is a key enzyme in the metabolism of phospholipids. Because a disordered phospholipid metabolism has frequently been reported in schizophrenia, we investigated the PLA2 activity in serum from 14 drug-free paranoid schizophrenic patients, 20 healthy controls, and 8 nonschizophrenic psychiatric patients. Schizophrenics showed significantly higher PLA2 activity than healthy controls and nonschizophrenic patients. The increment in schizophrenics was not due to increased concentration of pancreatic secretory PLA2, as concerning pancreatic PLA2 no differences were found among the 3 proband groups. The present findings confirm the results of our previous study and suggest that increased serum PLA2 activity might reflect an increment in the intracellular enzyme activity in schizophrenia. In the brain the activation of intracellular PLA2 results in changes in neuronal activity due to alterations in receptor sensitivity and in neurotransmitter metabolism. The possibility that such PLA2-induced mechanisms are involved in the pathogenesis of schizophrenia should be investigated in further experiments.

Adult↗

PubMed1990

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A2.

Hydrolysis by pancreatic and snake venom (Crotalus atrox) phospholipase A2 of fluorescent monolayers of pyrene-labelled phosphatidylglycerol on solid support was studied. We used a fluorescence microscope equipped with video camera, video recorder and an image analyzer to monitor changes in fluorescence. Decrease in pyrene excimer emission was evident when pyrene phosphatidylglycerol monolayers transferred onto quartz glass slides (at a surface pressure of 15 mN m-1) were subjected to enzymatic hydrolysis. Snake venom phospholipase A2 could hydrolyze the monolayers almost completely while pancreatic phospholipase A2 could cause only 50% decrease in fluorescence intensity. EDTA totally inhibited the action of both A2 phospholipases. When monolayers were transferred onto solid supports at a surface pressure of 31 mN m-1 C. atrox phospholipase A2 could still exert activity whereas porcine pancreatic phospholipase A2 was inactive.

Crotalid Venoms↗

PubMed1990

Pancreatic and catalytic phospholipase A2 in relation to pregnancy, labor and fetal outcome.

The serum pancreatic and catalytic phospholipase A2 level (PLA2) in human pregnancy is normal, and the increase of pancreatic enzyme before delivery is small. In patients with pruritus associated with obstetric hepatosis maternal serum had a slightly lower pancreatic PLA2 level if the cholic acid level was higher. Umbilical cord blood has twice as much pancreatic PLA2 as maternal blood. If the enzyme concentration was very high, pregnancies were postterm and the newborns had low Apgar scores. Amniotic fluid contains these enzymes, but there is no change in the enzymes during the course of pregnancy. The amount of pancreatic enzyme was not reflected in catalytic activity. A very high PLA2 activity was observed in 1 patient with suspected amniotic fluid embolism and in the meconium, but low in the first urine of neonates.

Amniotic Fluid↗

PubMed1989

Human pancreatic phospholipase A2 in acute necrotizing pancreatitis.

The activity and the content of phospholipase A2 (PLA2), a potential 'toxin' in pancreatitis, were determined separately by respective methods in pancreatic tissue resected from 22 patients treated for acute necrotizing pancreatitis. Correspondent enzyme assays were analyzed in the serum of 6 last patients. In cases with total necrosis in the tissue resected, the pancreatic PLA2 activity, but not the content, was almost totally lost. Serum PLA2 activity slightly decreased within the extension of pancreatic necrosis. The timing of sampling, number of positive Ranson signs or the course of the disease had no influence on the tissue PLA2 results. Serum PLA2 activity showed a correlation with tissue PLA2 activity.

Acute Disease↗

PubMed1988

A model for the molecular mechanism of interfacial activation of phospholipase A2 supporting the substrate theory.

Changes occurring in the activity of porcine pancreatic phospholipase A2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2-di[6-(pyren-1-yl)butanoyl]-sn-glycero-3-phosphocholine or 1-[6-(pyren-1-yl)butanoyl]-2-[6-(pyren-1-yl)hexanoyl]- sn-glycero-3- phosphocholine were studied. A 2-fold enhancement was observed in the activity of phospholipase A2 towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A2 activity at the critical micellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A2.

Animals↗

PubMed1988

Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate.

A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗