GLA Content Research Articles

Effect of gamma-linolenic acid on lipid metabolism in laying hens

1. 1. Single comb white leghorn laying hens were given diets with additional mould, Mucus circineloides, containing γ-linolenic acid (GLA) at levels of 0, 2.59 and 5.06 g GLA/kg diet ad lib. for 2 weeks and serum lipid contents were determined in experiment 1. 2. 2. Serum low density lipoprotein and chylomicron levels were significantly reduced with the increase of dietary GLA levels. Serum triglyceride and cholesterol tended to be lowered by dietary GLA, but not significantly different. 3. 3. Effects of mould GLA and extracted oil GLA on the egg yolk cholesterol concentration and fatty acid composition were compared in experiment 2. Both mould GLA and extracted oil GLA diets containing 5.32 and 4.71 g GLA/kg diet were given ad lib. for 2 weeks. 4. 4. Yolk cholesterol content was not affected by dietary GLA sources. Content of GLA in the yolk was not altered, although that of arachidonic acid was enhanced by dietary GLA supplementation, particularly by the extracted oil GLA. 5. 5. It is suggested that GLA is rapidly metabolized to arachidonic acid in the body and incorporated into the yolk.

Radiocarbon Dating of Bone Osteocalcin: Isolating and Characterizing a Non-Collagen Protein

Osteocalcin, a non-collagen bone-matrix protein, has been examined as a possible source of autochthonous14C data in fossil bones where collagen has been seriously degraded. Extraction procedures for osteocalcin yield a well-characterized product that can be clearly distinguished from collagen. The Gla content indicates that osteocalcin is present in the fossil bones at levels similar to the range present in modern bone. However, it appears to be extracted primarily as proteolytic polypeptide fragments rather than as an intact protein. Concordant14C determinations are obtained on osteocalcin and gelatin extracts from the same bone when the collagen is relatively well preserved. However, increasing discordances in the14C values of the osteocalcin and gelatin fractions are associated with reduced concentrations of the gelatin extract in the bone.

Determination of protein-bound urinary gamma-carboxyglutamic acid in calcium nephrolithiasis

Nephrocalcin is a urinary γ-carboxyglutamic acid (Gla) containing protein that may be a physiological inhibitor of calcium oxalate nephrolithiasis. Nephrocalcin isolated from urine of stone formers seems to be abnormal in lacking Gla that is required for inhibitory activity. In order to study this hypothesis, we compared the protein-bound urinary Gla contents in 32 calcium oxalate stone formers and in 24 controls. Protein-bound Gla was resolved by reversed-phase high-performance liquid chromatography after elimination of free Gla, alkaline hydrolysis and precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Protein-bound urinary Gla concentrations were similar in stone formers (0.83 ± 0.38 μmol/l, mean ± SD) and controls (0.81 ± 0.27) and were less than 5% of free urinary Gla. However, excretion rates of free and protein-bound Gla (nmol/min) were higher in stone formers ( P=0.006 and P=0.002). Positive correlations ( P=0.000) were observed between free and protein-bound Gla both in controls and in stone formers. These results do not support the hypothesis of a lacking Gla nephrocalcin in stone formers.

Isolation and some properties of 11- and 9-Gla prothrombins from normal plasma

The relationship of prothrombin structure to function with respect to gamma-carboxyglutamic acid (Gla) residues can be effectively evaluated by characterizing the behavior of prothrombin isomers differing in Gla content. In addition to the isolation of a whole spectrum of Gla-deficient, 0- to 9-Gla isomers from dicoumarol-treated plasma, prothrombin isomers containing 11 (10.90) and 9 (8.85) Gla residues have now been isolated from normal bovine plasma. The isomers were isolated by barium citrate adsorption, elution, and finally by heparin-agarose, DEAE-cellulose, and immuno-affinity chromatographies. Each of the purified isomers showed a single component by agar gel and sodium dodecyl sulfate - polyacrylamide gel electrophoresis. By agar gel electrophoresis, the 11-Gla prothrombin isomer moved the fastest, followed by the 10-, and lastly the 9-Gla isomer, independent of Ca2+. The corresponding 9-, 10-, and 11-Gla prothrombin fragments 1 exhibited similar migration tendencies. By gel electrofocusing, 11- and 9-Gla fragments 1, respectively, focused anodal and cathodal to 10-Gla fragment 1. The Ca2+-induced decrease in the intrinsic fluorescence in 11-, 10-, and 9-Gla fragments 1 was 48, 40, and 45%, respectively. This metal-induced structural change did not correlate with the functional, thrombin-generating property of the isomers, as the 9-Gla variant exhibited 75%, and the 11-Gla 110-115%, of normal coagulant activity.

Increase in Arachidonate Content of Tissue Lipids in Rats Fed with Mold Oil Containing γ -Linolenate

The dietary effects of mold oils from Mortierella ramanniana var. anglispora (IFO 8187), a lipid accumulative fungus with varying content of γ-linolenate (GLA), on plasma, liver and erythrocyte lipids in rats were examined using GLA at various concentrations and other conventional oils (palm, olive, safflower oil and tallow). The diet with mold oil caused higher arachidonate content in tissue lipids and the same growth as did the other dietary oils. A comparison of the mold oil with the control at similar (n-6) fatty acid content and comparison of mold oil with safflower oil diet indicated the possible acceleration of Δ6-desaturation by GLA and inhibition of Δ6-desaturation by high linoleate content. A dietary GLA content exceeding 10% caused decrease in the arachidonate content of tissue lipid in the rats.

Increase in the γ‐linolenic acid content by solvent winterization of fungal oil extracted fromMortierella genus

The fungal oil extracted fromMortierella ramanniana var.angulispora (IFO 8187) was solvent winterized in order to raise the content of γ‐linolenic acid (GLA). Effects of winterization conditions (solvent, oil concentration in the solvent and temperature) and changes of glyceride compositions were discussed. The fungal oil was separated into four diglycerides and 17 triglycerides (TG) with high performance liquid chromatography. The predominant species were POO, POP and LOP, whose contents were 24.4, 22.9 and 9.4% of the total TG, respectively. Ethanol at 4°C gave the highest GLA content of 10.5% in spite of lower yield than with acetone at −20°C. The highest separation efficiency for GLA (ηGLA) was 0.27 with acetone at −20°C and 10% oil concentration, resulting in 8.3% of GLA from the fungal oil at 5.7% LGA. In case of lower oil concentration at 5–20%, ηGLA showed higher in the following order: acetone (−20°C)>n‐hexane (−20°C)>acetone (4°C)>petroleum ether (−20°C). The winterization process also proved to be effective for the separation of TG type, Sa2U (Sa; saturated fatty acid; U, unsaturated fatty acid) into the crystallized fraction and SaU2 into the liquid fraction. Acetone at −20°C showed higher separation efficiency for triunsaturated TG than the other solvents.

Incorporation of linoleic acid and its conversion to λ‐linolenic acid in fungi

AbstractThe incorporation of [1‐14C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6‐desaturation of [1‐14C]LA. [1‐14C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1‐14C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1‐14C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1‐14C]LA into λ‐linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse‐chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1‐14C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.

Enzymatic fractionation of evening primrose oil by rape lipase: Enrichment of gamma-linolenic acid

Rape lipase discriminates strongly against γ-linolenic acid (allcis-6, 9, 12–18∶3; GLA). GLA in the fatty acids from evening primrose oil was concentrated from 9.5% to 62% with a 95% yield during esterification of these fatty acids with butanol catalyzed by rape lipase. Hydrolysis of evening primrose oil catalyzed by the rape lipase raised the GLA content in unhydrolyzed acylglycerols to 28%.

Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization

Isolation and characterization of gamma-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.

Studies on Production of Lipids in Fungi. XXII.

Nine fungi of Mortierella genus were cultivated batchwise in a 30L-fermenter in high con-centration glucose medium (100-200g/L). As a result, four strains attained a γ-linolenic acid (GLA) productivity exceeding 2g/L. The strain content of GLA in the fatty acids of the lipids obtained was from 6.6-7.8%. Using M. ramanniana var. angulispora IFO 8187, one of the four strains, the effects of various cultivation conditions on GLA productivity were investigated. Culti-vation at a higher content of GLA in the total lipids was generally noted in strains with lower lipid productivity, thus giving lower GLA yield. Consequently, cultivation to obtain high GLA yield is possible in a cultivation of high lipid productivity rather than high GLA content. Two types of repeated batch cultures with one and two fermenters, respectively, were made to obtain higher lipid productivity. A maximum lipid productivity of 13.9g/L/d was realized in the 2nd fermenter with a double stage repeated batch culture. GLA yield in the repeated batch culture increased with lipid productivity.

Osteocalcin

The vitamin K-dependent protein of bone, osteocalcin (bone Gla protein) is a specific product of the osteoblast. A small fraction of that synthesized does not accumulate in bone but is secreted directly into the circulation. Upon catabolism of osteocalcin, its characteristic amino acid, gamma-carboxyglutamic acid (Gla), is excreted into the urine. Both serum osteocalcin and urine Gla are currently being used for the clinical assessment of bone disease. The authors summarize the current understanding of the structure and function of osteocalcin in bone and evaluate the clinical studies done using serum osteocalcin and urinary Gla to monitor bone turnover. Factors that affect the measurement of osteocalcin concentrations in the blood are osteoblastic synthesis, content of Gla in the protein, drug-induced alterations in osteocalcin's affinity for bone, hormonal status, renal function, age, sex, timing of blood collection, and specificity of the radioimmunoassay. With these considerations, serum osteocalcin measurements provide a noninvasive specific marker of bone metabolism.

Monoclonal antibodies to prothrombin

Hybridoma technology was used for the production of murine monoclonal antibodies to bovine normal prothrombin. Hybrid cell cultures were assayed for the production of antibodies, both in the absence and presence of calcium ions, by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody-producing cell lines were cloned two times and grown as ascites tumors. Monoclonal antibodies (McAb), isolated by affinity chromatography (Protein A-Sepharose), were tested for their affinity for normal (10-Gla) and dicoumarol-induced abnormal prothrombins containing 2, 5, 7, 8 and 9 α-carboxyglutamyl (Gla) residues. A total of 24 McAb were obtained and the immunoglobulins were of the IgG1 subclass. Nine of the twenty-four McAb did not require Ca 2+ for the formation of Ag-Ab complexes, and reacted equally with normal and Gla-deficient prothrombins. These antibodies had affinity for prethrombin (PI) but not for the Gla-containing prothrombin fragment1 (Fl) portion of the molecule. In contrast, the 15 Ca 2+-dependent McAb reacted with F1 but not with P1. They discriminated the abnormal prothrombins based upon their Gla content. For example, though all the Ca 2+-dependent McAb formed Ag-Ab complexes with 9-, essentially none formed with 5- or less-Gla prothrombins. [Some reacted equally with 9- and 10-Gla (normal) prothrombin while others had only 25% of normal affinity for 9-Gla isomer]. Only four and twelve of the 15 McAb had some affinity for 7-and 8-Gla variants respectively. These results show that antibodies which react with the Ca 2+-stabilized conformation of prothrombin are not specific for normal prothrombin, as has been reported in the literature.

Fatty acid composition of Spirulina strains grown under various environmental conditions

The fatty acid distribution in 19 strains of Spirulina was studied. All but one contained γ-linolenic acid (GLA). No GLA was found in S. subsalsa, which had a very high content of palmitoleic acid. The fatty acid content of all but one of the tested strains increased with cultivation temperature and the relative amount of polyunsaturated fatty acid decreased. The highest content of GLA was found at 30–35° for most strains. High light intensities at a high temperature (38°), while not affecting the fatty acid composition, had a drastic effect on the fatty acid content, reducing it by as much as 46 %.

A colorimetric procedure for the determination of γ-carboxyglutamic acid

A colorimetric method for the determination of γ-carboxyglutamic acid (Gla) is presented. The procedure is based on the following results. (a) Gla is quantitatively converted into a proline derivative by reaction with acetaldehyde. (b) This derivative is spectrophotometrically detected by the secondary amine reagent: nitroprusside and acetaldehyde in alkaline medium. Under the reported conditions, Beer's law is obeyed for concentrations of Gla varying from 0.5 to 5 × 10 −4 m. The method has been used to determine the Gla content in urine samples.

1111 URINARY EXCRETION OF γ-CARBOXYGLUTAMIC ACID (GLA) IN X-LINKED HYPOPHOSPHATEMIA (XLH) AND AUTOSOMAL RECESSIVE VITAMIN D DEPENDENCY (ARVDD)

The mineralization defect in vitamin D deficiency (animal model) results in decreased turnover of osteocalcin, a unique vitamin K-dependent calcium-binding protein of bone matrix. GLA is the modified glutamate residue that is excreted quantitatively after osteocalcin degradation. To determine whether a similar effect occurs in mendelian rachitic disorders of man, we assayed urinary content of GLA. We studied 11 XLH patients (5M, 6F) treated with calcitriol (1,25-(OH)2D3) and phosphate. Urinary GLA excretion (50±15(SD) μmol/g creat) was not different from normal (44±11 μmol/g creat, n=63). We compared GLA excretion in 5 ARVDD patients in a vitamin D-depleted state with 7 ARVDD patients receiving a therapeutic dose of 1,25-(OH)2D3 (1.0-1.5 μg/day). GLA excretion in the D-depleted ARVDD group was significantly lower than normal (31±6 μmol/g creat, p=.006). GLA excretion was higher in the 1,25-(OH)2D3 treated group (44±4 μmol/g creat, p < .001) and not different from controls. These observations suggest that osteocalcin turnover is decreased in untreated individuals with impaired mineral metabolism. GLA excretion appears to be a useful marker for turnover of bone matrix in disorders of mineralization.

Atherocalcin, a γ-carboxyglutamic acid containing protein from atherosclerotic plaque

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.