Low vitamin D status may be involved in development of osteoporosis. Augmentation of vitamin D status by exposure to UV-B can not cause vitamin D toxicity, but carries the risk of skin cancer development. Oral vitamin D supplementation of postmenopausal women living at high latitudes in winter is therefore recommended. There is concern that this strategy may cause vitamin D toxicity. Little is known about the buffering role of adipose tissue in vitamin D storage and release. The aim of this study was to investigate vitamin D storage in rat adipose tissue, and its subsequent unstimulated and stimulated release. Female Wistar rats were supplemented with 1500 IU vitamin D3/day during 14 days. Plasma vitamin D3 and 25-hydroxyvitamin D [25(OH)D], and adipose tissue vitamin D3 were monitored during 99 days. From day 14 a subgroup of rats was fasted during 72 hours to investigate the effect of stimulated vitamin D release from adipose tissue. Following vitamin D supplementation, plasma vitamin D3 reached steady state levels within 3 days. 25(OH)D increased more slowly to reach plateau levels from about day 10. Adipose tissue vitamin D3 rose linearly until day 14. Following discontinuation of vitamin D3 supplementation, plasma vitamin D3 decreased more quickly (t1/2=29 hours) than 25(OH)D (t1/2=18 days). There were no changes in adipose tissue vitamin D3 contents. Fasted rats lost about 10% weight, but did not show different courses of plasma vitamin D3 and 25(OH)D, compared with ad libitum fed counterparts. Their adipose vitamin D3 content (in nmol/g wet weight) rose, indicating that fatty acid mobilization from adipose tissue occurs more easily than that of vitamin D. We conclude that orally supplemented vitamin D rapidly accumulates in adipose tissue, but slowly releases following discontinuation. Storage capacity seems unsaturable and fasting does not cause its massive release. Uptake of vitamin D in adipose tissue may be an important factor in the prevention of vitamin D toxicity and maintenance of long term adequate vitamin D status. Lipiden

Lipoprotein(a) (Lp(a)) is a LDL-like particle with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) is highly homologous to parts of the plasminogen molecule, and numerous investigations have shown interference of Lp(a) with functions of plasminogen. In this report we studied the influence of apo(a) phenotype on the binding of Lp(a) to plasmin-modified immobilized des-AA-fibrinogen (desafib-X).Results indicate that Lp(a) binds to desafib-X in a specific and saturable way. There was a strongly significant negative correlation between apo(a) isoform length and maximal number of Lp(a) particles bound to the desafib-X matrix (N=18, r=0.84, p=0.002). There was no relation between apo(a) isoform length and K-d for the binding of Lp(a) to desafib-X. In two donors (10%) no specific binding to desafib-X was observed, as well as to lysine-sepharose.In conclusion, apo(a) isoform length influences the amount of Lp(a) binding to desafib-X. This implies that small isoforms apart from their usual higher plasma Lp(a) concentrations also are potentially more thrombogenic. The fact that Lp(a) from two donors did not bind to desafib-X, suggests that mutations may exist in the K4-10 domain of apo(a) abolishing its lysine-binding ability.

Human plasma Lp(a) is susceptible to various sulfhydryl compounds. In this study we present evidence indicating that after treatment of Lp(a) with sulfhydryl compounds, immunoreactivity is changed, structural changes occur and functional characteristics regarding the numerous kringle structures in apo(a) disappear. Purified Lp(a) was subjected to variable concentrations (0.01-10 mM) of various sulfhydryl compounds: DTT, 2-mercapto-ethanol (BME), N-acetylcysteine (NAC) and homocysteine (HCys). Free SH groups were blocked by iodoacetamide. Reduced and alkylated Lp(a) was tested in two ELISAs, one detecting apo(a) alone and one detecting apo(a)-apoB complexes. In both ELISAs polyclonal antibodies were used. For comparison a commercial apo(a) IRMA utilizing two monoclonal antibodies was used. The results indicate that a similar decrease in response of both ELISAs is observed, whereas the IRMA response is less affected. Western blotting of "DTT treated" Lp(a) after SDS-PAGE under nonreducing conditions showed that separate apo(a) and apoB-100 bands became detectable at 1 mM DTT. Native PAGE (2.5-16%) indicated structural changes of Lp(a) beginning to occur at 0.03 mM DTT. Epsilon-aminocaproic acid-inhibitable binding of "DTT-treated" Lp(a) to Desafib-X decreased with increasing DTT concentrations in concert with a loss of the capacity of Lp(a) to inhibit plasminogen activation upon treatment with DTT. The observed immunological and functional changes of Lp(a) indicate that apo(a) kringle function is severely affected by sulfhydryl compounds.

Three commercially available assays (an enzyme-linked immunosorbent assay ELISA, an immunoradiometric assay, IRMA, and a nephelometric assay) for the determination of lipoprotein(a) [Lp(a)] were compared with respect to the dependency of these assays on the various apolipoprotein(a) [apo(a)] isoforms. Although there was a strong correlation between the three methods, a significant difference between the absolute values (mg/L) was observed (p < 0.001). Using purified Lp(a) preparations, we showed that the ELISA assay quantifies the Lp(a) concentration on a molar basis, independently of the apo(a) isoform size. The IRMA and the nephelometric assay however are apo(a) isoform size dependent and overestimate the Lp(a) concentration of large apo(a) isoforms whereas the amount of small apo(a) isoforms is underestimated. In general, the isoform dependency of the Lp(a) quantification is of limited clinical relevance. In this study, inconsistent risk assignments are made in approximately 3% of the cases, when the Lp(a) concentrations obtained with the apo(a) isoform dependent assays are compared with the isoform independent ELISA.

Summary Lipoprotein(a) [Lp(a)] is recognized as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) has a high homology with plasminogen (Pg). In vivo, Pg is activated on a fibrin surface by tissue Pg activator (tPA). We prepared Lp(a) from plasma by sequential ultracentrifugation followed by lysine-sepharose affinity chromatography. We found that a changing (donor dependent) fraction of the Lp(a) did not bind to lysine-sepharose. This fraction, designated Lp(a)lys–, was further purified using gel filtration. Bound Lp(a) [Lp(a)lys+] was eluted with 0.2 M EACA. Apo(a) isoforms in both fractions were identical. In contrast Lp(a)lys+ inhibited Pg activation by tPA in vitro (IC50% 20 mg/1), whereas Lp(a)lys– did not. In addition Lp(a)lys– did not bind to CNBr-digested fibrinogen whereas Lp(a)lys+ did (K d, app = 0.2 nM). Therefore we conclude that a changing donor dependent fraction of human plasma Lp(a) does not inhibit Pg activation in vitro and does not bind to CNBr-digested fibrinogen.

Lipoprotein (a) (Lp(a)) is recognised as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional protein, apo(a), bound to the apolipoprotein B-100. This apo(a) has a high homology with plasminogen (Pg). This prompted us to investigate The possible interference of Lp(a) with Pg activation in vitro. We purified Lp(a) with sequential ultracentrifugation followed by affinity chromatography using lysine-sepharose. Different concentrations of Lp(a) were added to a mixture of Pg, t-PA and CNBr-digested fibrinogen. The plasmin formation was quantified with a plasmin specific substrate S-2251. The experiments indicate that Lp(a) inhibits the rate of plasmin formation in a concentration dependent way with an uncompetitive inhibition constant Kiu = 38 nM. Experiments with varying amounts of fibrinogen fragments show that these fragments are imperative for the inhibition of the Pg activation by Lp(a). Excess of these fragments attenuates the inhibition. This suggests that Lp(a) inhibits Pg activation through the formation of a Lp(a)-fibrin(ogen) complex.