Pekka H. Mäenpää

Serum fatty acids, apolipoproteins, selenium and vitamin antioxidants and the risk of death from coronary artery disease.

The independent association of serum concentrations of saturated and polyunsaturated fatty acids, apolipoproteins AI and B, selenium and vitamins A and E with the risk of death from coronary artery disease (CAD) was studied in 92 persons with no previous myocardial infarction, who died from CAD during a 5-year follow-up, and their 92 1-to-1 matched controls. Case-control pairs came from a randomly drawn population sample of approximately 12,000 persons aged 30 to 64 years from 2 provinces of eastern Finland, an area with exceptionally high CAD mortality. Control subjects were matched for sex, age, serum cholesterol, mean arterial pressure, tobacco consumption and history of cardiovascular diseases. The persons who died of CAD had lower serum esterified arachidonic acid concentrations before follow-up than the control subjects (41 vs 48 mg/liter, p = 0.05), and this difference was greater for pairs with no chest pain on effort (36 vs 50 mg/liter, p less than 0.05). The adjusted risk of CAD death in persons with a serum polyunsaturated to saturated (P/S) fatty acid ratio of 0.28 or less (in the lowest tertile) was 3.5-fold (95% confidence interval [CI], 1.5 to 8.2) compared with those with higher serum P/s ratios in a multivariate logistic model and 5.6-fold (95% CI 1.6 to 19.8) for pairs with no chest pain on effort. A low serum apolipoprotein AI concentration (1.25 g/liter or less, in the lowest tertile) was associated with a 2.5-fold (95% CI 1.1 to 5.7) adjusted risk of CAD death among the chest pain-free persons.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver Adenine Nuldeotides: Fructose-Induced Depletion and Its Effect on Protein Synthesis

Intravenous administration of D-fructose to rats rapidly depletes liver adenosine triphosphate and inorganic phosphate; marked elevations of uric acid and allantoin in plasma follow. Concomitantly the incorporation of DL-leucine-1-14C into liver protein is almost completely inhibited.

Relationship of serum selenium and antioxidants to plasma lipoproteins, platelet aggregability and prevalent ischaemic heart disease in Eastern Finnish men

In a cross-sectional population study of 1132 unselected Eastern Finnish men aged 54 years, serum selenium concentration had a weak positive association with plasma HDL cholesterol (standardised partial regression coefficient, beta = 0.061, P = 0.019) and a fairly strong inverse relationship (beta = -0.223, P less than 0.001) with the extent of ADP-induced platelet aggregation. Neither plasma ascorbate concentration nor alpha-tocopherol to total cholesterol ratio had any association with plasma lipoproteins, platelet aggregability or prevalent ischaemic heart disease (IHD). When a covariance-correction was applied, men with ischaemic ECG findings at exercise had a lower mean serum selenium than others (81.5 micrograms/l vs. 85.9 micrograms/l, P less than 0.01 for difference). This difference was equally large for men with neither symptoms nor previous diagnosis of IHD.

Cross-linking of Osteopontin by Tissue Transglutaminase Increases Its Collagen Binding Properties

Osteopontin, a major noncollagenous bone protein, is an in vitro and in vivo substrate of tissue transglutaminase, which catalyzes formation of cross-linked protein aggregates. The roles of the enzyme and the polymeric osteopontin are presently not fully understood. In this study we provide evidence that transglutaminase treatment significantly increases the binding of osteopontin to collagen. This was tested with an enzyme-linked immunosorbent assay. The results also show that this increased interaction is clearly calcium-dependent and specific to osteopontin. In dot blot overlay assay 1 μg of collagen type I was able to bind 420 ng of in vitro prepared and purified polymeric osteopontin and only 83 ng of monomeric osteopontin, indicating that the transglutaminase treatment introduces a 5-fold amount of osteopontin onto collagen. Assays using a reversed situation showed that the collagen binding of the polymeric form of osteopontin appears to be dependent on its conformation in solution. Circular dichroism analysis of monomeric and polymeric osteopontin indicated that transglutaminase treatment induces a conformational change in osteopontin, probably exposing motives relevant to its interactions with other extracellular molecules. This altered collagen binding property of osteopontin may have relevance to its biological functions in tissue repair, bone remodeling, and collagen fibrillogenesis.

Depletion of liver adenine nucleotides induced by d-fructose: Dose-dependence and specificity of the fructose effect

Abstract The effect of d -fructose on adenine nucleotide metabolism was studied in rats in vivo by analyzing liver metabolite levels after intravenous fructose injections, and in vitro by measuring allantoin production by the isolated perfused liver after the addition of fructose to the perfusion medium. Significant depression of liver ATP and inorganic phosphate levels was demonstrated after fructose administration. Minimum effective dose was 0.25 m-mole, and maximum depression was observed after 5 min. Analogous, but quantitatively less impressive alterations were produced by l -sorbose and d -sorbitol, whereas d -glucose, d -galactose, d -mannose, d -ribose and 2-deoxy- d -glucose were without effect. In the perfusion studies, marked increase in allantoin production was observed after the addition of d -fructose to the perfusion medium. The minimum effective dose was 0.25 m-mole, corresponding to a mean initial concentration of 4.5 mM in the medium. Allopurinol prevented both the basal allantoin production and the fructose-induced increase. d -galactose, d -ribose, d -sorbitol, sodium- dl -lactate or ethanol did not influence the basal rate of allantoin release. The loss of liver adenine nucleotides in the in vivo experiments was of a similar order of magnitude as the amount of allantoin produced in the perfusion experiments, when calculated per total liver after equal doses of fructose. Therefore, accelerated degradation of preformed purines, rather than increased de novo synthesis, appears to be the main mechanism of fructose-induced increase in uric acid and allantoin production.

Early postmenopausal bone loss is associated with PvuII estrogen receptor gene polymorphism in finnish women : Effect of hormone replacement therapy

Genetic factors regulate bone mineral density (BMD) and possibly the development of osteoporosis. An association between estrogen receptor (ER) polymorphism, BMD, and postmenopausal hormone replacement therapy (HRT) has not been established. Therefore, we studied the influence of the ER genotype on BMD before and after a 5‐year HRT in a placebo‐controlled, population‐based, randomized group of 322 early postmenopausal women. The participants were randomized into two treatment groups: the HRT group (n = 145) received a sequential combination of 2 mg estradiol valerate and 1 mg CPA with or without vitamin D3, 100–300 IU + 500 mg calcium lactate/day (equal to 93 mg Ca2+), and the non‐HRT group (n = 177) received calcium lactate, 500 mg alone or in combination with vitamin D3, 100–300 IU/day. PvuII restriction fragment length polymorphism (RFLP) of the ERα was determined using polymerase chain reaction (PCR). BMDs of the lumbar spine (L2–4) and proximal femur were measured by using dual‐energy X‐ray absorptiometry (DXA). At the baseline, there were no significant differences in the lumbar or femoral neck BMDs between the three ER PvuII genotype groups (PP,Pp,pp). After 5 years, the BMD of the femoral neck remained unaltered and that of the lumbar spine increased by 1.7% in the HRT group, whereas both BMDs were decreased by 4–5% in the non‐HRT group. The ER genotype did not modulate the femoral neck BMD change during the follow‐up. In contrast, in the non‐HRT‐group the lumbar spine BMD decreased more in subjects with the ER genotypes PP (6.4%) and Pp (5.2%) than in subjects with the pp genotype (2.9%) (p = 0.002). In the HRT group, the relative changes of the lumbar spine BMD were similar in all three ER genotype groups. Thus without HRT, the pp genotype was associated with a smaller decrease in the lumbar spine BMD than the Pp and PP genotypes. Long‐term HRT seemed to eliminate the ER genotype‐related differences in the BMD. We conclude that subjects with the ER PvuII genotypes PP and Pp may have a greater risk of relatively fast bone loss after menopause than those with the pp genotype and that they may preferentially derive benefit from HRT. (J Bone Miner Res 2000;15:315–321)

Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells

Hormone-dependent accumulation of specific binding sites for 1,25(OH)2D3 and changes in human 1,25-dihydroxy-vitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH)2D3 were quantified by incubating cultured intact cells with [3H]1,25(OH)2D3 at 37 degrees C. Based on the uptake of 1,25(OH)2D3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH)2D3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH)2D3, and maximal binding was achieved with 0.2-0.7 nM 1,25(OH)2D3. Treatment with 1,25(OH)2D3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH)2D3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH)2D3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH)2D3.

The Protective Effect of Hormone‐Replacement Therapy on Fracture Risk Is Modulated by Estrogen Receptor α Genotype in Early Postmenopausal Women

Genetic factors regulate bone mineral density (BMD) and possibly development of osteoporosis. It has been suggested that estrogen receptor α (ERα) genotype is associated with BMD, but the association between ERα genotype, fracture risk, and postmenopausal hormone replacement therapy (HRT) has not been studied. Therefore, we evaluated whether ERα polymorphism is associated with fracture risk in a 5‐year trial with HRT in a population‐based, randomized group of 331 early postmenopausal women. The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg of estradiol valerate (E2Val) and 1 mg of cyproterone acetate with or without vitamin D3, 100‐300 IU + 93 mg calcium as lactate per day; and the non‐HRT group (n = 180) received 93 mg of calcium alone or in combination with vitamin D3, 100‐300 IU/day. All new symptomatic, radiographically defined fractures were recorded. Pvu II restriction fragment length polymorphism of the ERα was determined using polymerase chain reaction (PCR). In all, 28 women sustained 33 fractures during the approximately 5.1‐year follow‐up. In the HRT group, the ERα genotype (PP, Pp, and pp) was not significantly associated with fracture risk (p = 0.138; Cox proportional hazards model). When the genotype was dichotomized (PP + Pp vs. pp), the incidence of new fractures in the HRT group was significantly reduced in women with the P allele (p = 0.046) with the relative risk (HR) of 0.25 (95% CI, 0.07‐0.98), in comparison with the non‐P allele group. After adjustment for time since menopause and previous fracture, the association between the dichotomous genotype and fracture risk persisted with HR of 0.24 (95% CI, 0.06‐0.95; p = 0.042). In the non‐HRT group, the ERα genotype was not significantly associated with fracture risk. During HRT, women with the pp genotype have a greater fracture risk than those with the P allele. The results suggest that the pp genotype is a relatively hormone‐insensitive genotype, and it appears that women with the P allele may benefit more from the protective effect of HRT on fracture risk than women with the pp genotype.

Transglutaminase-catalyzed Cross-linking of Osteopontin Is Inhibited by Osteocalcin

Osteocalcin, the most abundant noncollagenous protein of bone matrix, has been demonstrated to inhibit bone growth by gene knockout experiments (Ducy, P., Desbois, C., Boyce, B., Pinero, G., Story, B., Dunstan, C., Smith, E., Bonadio, J., Goldstein, S., Gundberg, C., Bradley, A., and Karsenty, G. (1996)Nature 382, 448–452). Its specific functional mechanism in bone metabolism is, however, largely unknown. In this study, we provide evidence that osteocalcin has an inhibitory effect on tissue transglutaminase activity, as measured by cross-linking of osteopontin, another bone matrix protein. Using a set of synthetic peptides, we found that the inhibitory activity resided within the first 13 N-terminal amino acid residues of osteocalcin. An N-terminal peptide also inhibited cross-linking of another tissue transglutaminase substrate, β-casein. The inhibitory peptide was shown to have affinity for the substrates of transglutaminase rather than for the enzyme. Since the N terminus of osteocalcin exhibits homology to the substrate recognition site sequences of two transglutaminases, we conclude that the inhibitory effect is most likely due to competition with the enzyme for the transglutaminase-binding region of the substrates, osteopontin and β-casein, which prevents access of the enzyme to them to perform its function. The interference of osteocalcin with osteopontin cross-linking gives osteocalcin a new potential function as the first protein inhibitor of tissue transglutaminase. This suggests a specific role and a plausible mechanism for it as a modulator of maturation, stabilization, and calcification of bone matrix.

EB 1089, a novel vitamin D Analog with strong antiproliferative and differentiation-inducing effects on target cells

The physiologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3, plays an important role not only in the establishment and maintenance of calcium metabolism, but also in regulating cell growth and differentiation. As the clinical usefulness of 1alpha,25-dihydroxyvitamin D3 is limited by its tendency to cause hypercalcemia, new analogs with a better therapeutic profile have been synthesized. One of these new synthetic vitamin D analogs is EB 1089, which is characterized by an altered side chain structure featuring 26,27-dimethyl groups and two double bonds. This analog has been shown to be more potent than 1,25-dihydroxyvitamin D3 in inhibiting proliferation, stimulating differentiation, and inducing apoptosis in a number of different cell types, including cancer cells. Despite being more potent than 1alpha,25-dihydroxyvitamin D3 with respect to its cell regulatory effects, EB 1089 displays weaker calcemic side-effects. These characteristics make EB 1089 a potentially useful compound for the treatment of a diversity of clinical disorders, including cancer and metabolic bone diseases. A promising phase I study with EB 1089 in patients with advanced breast and colon cancer has already been carried out, and more clinical trials evaluating the clinical effectiveness of EB 1089 in other types of cancer are in progress.