Søren Risom Kristensen

A Common Methylenetetrahydrofolate Reductase (C677T) Polymorphism Is Associated With Low Bone Mineral Density and Increased Fracture Incidence After Menopause: Longitudinal Data From the Danish Osteoporosis Prevention Study

A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) has recently been associated with bone mineral density (BMD) in postmenopausal Japanese women. It is not known whether this effect is also present in European populations and whether it is caused by lower peak bone mass or accelerated postmenopausal bone loss. MTHFR genotyping was done in 1748 healthy postmenopausal Danish women participating in a prospective study of risk factors for osteoporosis. At the time of enrollment, 3–24 months after last menstrual period, the less prevalent genotype (TT, 8.7% of the population) was associated with significantly lower BMD at the femoral neck (ANOVA, p < 0.05), total hip (p < 0.01), and spine (p < 0.05 adjusted for lifestyle covariates, p = 0.06 without adjustment). The mean difference was between 0.1 and 0.3 SD, depending on measurement site. MTHFR genotype added significantly to prediction of BMD by weight and age. Fracture incidence was increased more than 2‐fold in subjects with the TT genotype (risk ratio [RR], 2.6; 95% CI 1.2–5.6). This remained significant when the Cox analysis was controlled for BMD (RR, 2.4; 95% CI 1.1–5.2). No differences in serum osteocalcin, bone‐specific alkaline phosphatase, and 25‐OH‐vitamin D were found between genotypes. The response to hormone replacement therapy (HRT) did not differ, but the association of the TT genotype with reduced BMD was maintained at the total hip after 5 years of HRT. The MTHFR TT genotype is associated with low BMD and increased fracture incidence in early postmenopausal women.

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.

Reference values for six enzymes in plasma from newborns and women at delivery

We have determined the distribution in cord blood from healthy newborns of six enzymes: creatine kinase, lactate dehydrogenase, aspartate and alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase. The concentration of enzymes were determined according to the methods recommended by the Scandinavian Committee on Enzymes. The distribution of isoenzymes and of enzymes in blood from women at delivery was investigated also. All distributions were positively skewed. The upper reference limits of cord blood exceeded those found in mother blood by a factor of eight for gamma-glutamyltransferase, and for lactate dehydrogenase and creatine kinase by a factor of two.

Importance of various types of metabolic inhibition for cell damage caused by direct membrane damage

Cell damage is caused by energy depletion or by direct membrane damage, or a combination when a direct membrane damage affects energy depleted cells. In this report it was investigated whether the extent of direct membrane damage induced by lysophosphatidyl choline (LPC) or phospholipase C (PhC) on quiescent fibroblasts depended on the metabolic state of the cells. When glycolysis was inhibited cell damage was always extensively increased, whereas cell damage was also increased to a minor degree when exposed to PhC during sole inhibition of oxidative phosphorylation. Acceleration of glycolysis in cells with a low rate of glycolysis resulted in a dramatic improvement of the membrane susceptibility within a few minutes. Thus, susceptibility of the cell membrane to direct membrane damage depends on the metabolic state. The results also emphasize previous findings that glycolysis has a special role in maintaining membrane function and integrity.

Quiescent fibroblasts in a cellular model system

Quiescent fibroblasts are non-dividing cells in a reversible postmitotic state induced by lowering the serum concentration of the medium (e.g. from 10% to 0.3%). Three to seven days after lowering the serum concentration only minor metabolic changes will take place in the cells. During this period the quiescent fibroblasts can be used experimentally in a model system for various periods of time.

Removal of calcium overload caused by A23187 is more dependent on glycolysis than oxidative phosphorylation

An increase of cytosol Ca2+ may be the mediator of irreversible cell damage after ATP depletion. Ca2+ influx can also be induced by addition of the ionophore A23187. The extent of cell damage caused by A23187 during concomitant metabolic inhibition was investigated on quiescent fibroblasts. Inhibition of glycolysis resulted in increased cell damage compared to control, whereas an increased rate of glycolysis during inhibition of oxidative phosphorylation attenuated cell damage. These results indicate that glycolysis plays an important part in the removal of entering Ca2+.

The influence of extracellular magnesium on cell damage induced by ATP depletion in human fibroblasts

Magnesium is an essential ion for the structural and functional integrity of cells. We have examined whether a low extracellular magnesium concentration contributes to cell damage induced by ATP depletion in a model system of quiescent fibroblasts. Cell damage assessed by the release of lactate dehydrogenase was slightly, but significantly increased in the absence of Mg2+. The increased cell damage may be caused by an increased Mg2+ loss during ATP depletion, by an altered cell membrane permeability for potassium and sodium, or by a disturbed balance between Mg2+ and Ca2+.

Dependency on Infarct Size Limits the Clinical Applicability of Non-Invasive Reperfusion Assessment by Biochemical Markers in Acute Myocardial Infarction

Background: Thrombolytic therapy often fails to obtain reperfusion in acute myocardial infarction (AMI). To allow further intervention, various methods for non-invasive reperfusion assessment, e.g. release kinetics of biochemical markers and ST segment recovery analysis on the ECG, have been proposed. Methods and Results: In 85 thrombolysed AMI patients <76 years with significant ST elevation on ECG and time delay ≤6 h, blood was drawn and ECGs recorded before and 90 min after the start of treatment. Myoglobin, creatine kinase-MB and troponin I were measured, and slopesand relative increases (RIs) of the markers were calculated. Moreover, the relative decrease in total sum of ST elevation (ST recovery0–90) on the ECG was calculated. A slope0–90 or RI0–90 below previously validated threshold values or an ST recovery <25% was considered indicative of failed reperfusion. Final infarct size was determined as cumulative release of LD1. Patients were followed for 1 year to register deaths, re-infarctions, and coronary revascularisation. The various non-invasive reperfusion indexes showed poor agreement, and no significant prognostic information was obtained. A positive correlation between increase rates and infarct size probably reflected a direct, concentration-dependent enhancement of release of biochemical markers in blood. Thereby, high-risk patients with large, non-reperfused infarcts might have been overlooked while only low-risk patients with small non-reperfused infarcts were recognised. This could readily explain the lack of correlation with long-term outcome. Conclusions: Dependency on infarct size seems to limit the usefulness of biochemical markers for reperfusion assessment. Correction for expected infarct size might improve performance in the future. At present, routine use of biochemical markers for reperfusion assessment is not justified.

Clinical usefulness of a functional test of neutralising streptokinase antibodies for the prediction of the thrombolytic effect of streptokinase in acute myocardial infarction.

We investigated whether the occurrence of neutralising streptokinase (SK) antibodies, as assessed by a new bedside test, was related to the effect of SK in acute myocardial infarction (AMI), assessed with continuous vectorcardiography (VCG). Twenty-eight patients with ST elevation, admitted ≤ 6 h after the onset of symptoms, treated with 1.5 MIU SK were included. The level of SK antibodies was assessed by the TAS Streptokinase Test Panel, measuring the lysis time of clotted fibrin in the sample. Clot lysis time was negatively correlated to ST vector magnitude (ST-VM) recovery at 90 min (rS = –0.40, p = 0.03), and clot lysis time was positively correlated to time to reperfusion, (rS = 0.42, p = 0.03). The predictive values of the test, however, were low. In conclusion, as the prediction of reperfusion in AMI after SK by the new test was poor, we cannot recommend it for clinical decision-making prior to the choice of the thrombolytic agent.