Menard M. Gertler

The Assessment of Insulin, Glucose and Lipids in Ischemic Thrombotic Cerebrovascular Disease

Sixty-one male patients with ischemic thrombotic cerebrovascular disease (ITCVD) and 61 age-matched controls were studied to determine the interrelationships between the primary risk factors of ITCVD. Impaired carbohydrate metabolism in ITCVD was reflected in the significantly greater number of abnormal oral glucose tolerance tests (GTT) and type IV lipoprotein abnormalities when compared to controls. Elevated uric acid and triglyceride levels also were observed in ITCVD. Glucose and immunoreactive insulin (IRI) response curves in ITCVD were elevated and exhibited delayed peaks despite the normal or abnormal classification of GTT, indicating that insulin is ineffective in restoring glucose to normal levels. In the ITCVD and control groups with abnormal GTT the free fatty acid (FFA) levels were elevated at fasting and lacked the characteristic rebound at two hours observed in subjects with normal GTT, supporting the theory of a glucose-FFA cycle and its role in carbohydrate disturbances.

Ischemic Heart Disease: Insulin, Carbohydrate, and Lipid Interrelationships

This study evaluates interrelationships between carbohydrate and lipid metabolism during oral glucose tolerance tests (GTT) in 65 ischemic heart disease (IHD) males and 69 age-matched healthy controls (age range 45 to 69 years). The frequency of abnormal GTT, usually accompanied by type IV hyperlipoproteinemia, was significantly higher in IHD (37%) than in controls (19%). The mean immunoreactive insulin (IRI) response curve of IHD patients with abnormal GTT showed an elevated and delayed peak at 2 hours. The mean free fatty acid response curve of IHD patients had a significantly lower rebound at 3 hours. IHD patients and controls with abnormal GTT showed significantly higher and lagging lactate levels at 2 and 3 hours. Incidence of abnormal GTT was neither related to relative weight nor to elapsed time from onset of IHD to time of examination. Canonical correlations revealed that IRI is the common denominator in both carbohydrate and lipid abnormalities in IHD.

Oxidative phosphorylation studies in normal and experimentally produced congestive heart failure in guinea pigs: a comparison.

As one tries to formulate a biochemical approach to the heart in failure or to the effect of certain drugs such as digitoxin, one is intrigued by an observation made many years ago with the heart-lung preparation. FIGURE 1, from Evans’, shows the effect of increasing ventricular pressure on oxygen consumption and the efficiency of output by the heart. At first (curve 1) the diastolic volume and the oxygen consumption (curve 2) show a parallel rise. With increasing intraventricular pressure, however, the diastolic volume begins to decline. The interesting fact is that while the diastolic volume is declining the oxygen consumption continues to rise in almost linear fashion. Therefore it appears that beyond a certain ventricular pressure the efficiency of substrate utilization is decreased. At higher pressures there seems to be a decrease in the efficiency of transfer of utilizable energy to the muscle. If a heart-lung preparation in this state is treated with digitalis, there is a rise in diastolic volume (curve 3), while oxygen consumption (curve 4) remains about the same. From this curve it would appear that, in biochemical terms, digitalis is, “recoupling” a system that is “uncoupled.” With these considerations in mind, we looked for experimental techniques that would make it possible to approach the problem of failure and the effect of certain drugs, such as, digitalis, on a biochemical basis. It seemed to us that there would be three general areas where one could look for these effects in the heart: first, the energy-generating system (oxidative phosphorylation) ; second, an energy-transferring system, such as, myokinase and nucleoside diphosphokinase; and, third, the energy-utilizing system, for example, actomyosin. In the initial experiments2 the yield of oxidative phosphorylation of cardiac mitochondria from digitalized, normal guinea pigs was compared to that of normal untreated animals (TABLE 1). In these experiments we used three substrates, one of which was a-ketoglutarate with a malonate block; this yields a one-step oxidation of a-ketoglutarate to succinate. We compared mitochondria from the normal and the digitalized animals and found practically no difference. Note also that the oxygen uptake is almost the same in the treated and the untreated animals. The same situation prevails with glutamic acid and with acetate as substrates. Thus it would appear that digitalization of the normal guinea pig does not lead to a change in the efficiency of oxidative phosphorylation and has no effect on the rate of substrate oxidation. I t seemed possible to us that the effect of digitalis might show up in mitochondria from animals in congestive heart failure. However, in order to perform such experiments, it was necessary first to devise a method for pro-

Differences in efficiency of energy transfer in mitochondrial systems derived from normal and failing hearts.

Summary and Conclusions 1. Oxidation rates employing a-ketoglutarate as substrate and efficiency of phosphorylation coupled with oxidation were studied in heart mitochondrial systems prepared from normal guinea pig hearts and hearts from guinea pigs in congestive heart failure. 2. ON O2 values were 1170 and 1032 for the normal guinea pig hearts and experimental guinea pig heart mitochondrial systems for α-ketoglutarate oxidation respectively. P/O ratios were 2.95 and 2.91 respectively. 3. Addition of cofactors (CoA, DPN and TPN), increased ON O2 values from 1170 to 1480 in the normal heart mitochondrial systems and from 1032 to 1524 in the mitochondrial systems derived from animals in experimental congestive heart failure. P/O ratios decreased slightly in the normal systems (2.95 and 2.32) while the P/O ratio decreased from 2.91 to 1.95 in the animals in congestive heart failure. 4. The increase in energy production parallels the overall increase in ∼ phosphorus during addition of cofactors in both normal animals and failure animals. Although the efficiency of phosphorylation in the mitochondrial system derived from failing hearts is markedly decreased during the influence of the cofactors, energy production is maintained by the inordinate increase in respiration. The possible relationship of this observation to basic physiologic mechanisms is suggested.

Neutralization of heparin by histone and its subfractions

The neutralization of heparin by histone and its subfractions has been systematically studied by measuring the effect of heparin on the esterolytic and proteolytic activity of thrombin. These results were compared with protamine sulfate, a most commonly used heparin-neutralizing agent. This study reveals that potencies of different fractions of histone are not similar. The antiheparin potency is in the order: lysine-rich histone greater than crude histone greater than arginine-rich histone. Histone binds strongly to heparin - Sepharose gel. The ability of histone to bind heparin can be utilized to fractionate heparin. By affinity chromatography on histone - Sepharose gel commercial heparin has been fractionated into components having a wide range of anticoagulant activities. The highest activity fraction, eluted around 1.0 NaCl, has 66% higher anticoagulant activity than the commercial heparin used.

Production of experimental congestive heart failure in the guinea pig.

Summary and conclusion An operative technic has been described which causes development of congestive heart failure in the guinea pig within 8 to 12 days. Essentially the technic involves intrathoracic incision and restriction of aortic blood flow by ligature surrounding the aorta. Tissues derived from this preparation may be subjected to biochemical and enzymologic studies because of feasibility of preparing slices, homogenates, cell free suspensions and isolated particles. Preliminary biochemical work has been accomplished with this preparation.

The catalytic activity and physical properties of bovine thrombin in the presence of dimethyl sulfoxide

Dimethyl sulfoxide produces an opposite effect on the esterase and amidase activities of bovine thrombin. The esterase activity is increased by two fold but the amidase activity is decreased to 9% of the initial activity in 20% dimethyl sulfoxide. The stimulation of the esterase activity is due to the change in Vmax rather than Km for the substrate p-Tosyl-L-Arginine methyl ester. The inhibition of the esterase activity of thrombin by NaCl is not affected due to the addition of dimethyl sulfoxide. Ki for NaCl, 0.03 M, is the same for both in the absence and in the presence of 10% dimethyl sulfoxide. The catalytic activity of thrombin is inhibited by heparin. This effect is significantly decreased by dimethyl sulfoxide. The dissociation constant of heparin-thrombin complex, measured in the absence and in the presence of 10% dimethyl sulfoxide are 4 nM and 28 nM respectively. Thermal stability of thrombin, determined by monitoring catalytic activity, is increased in the presence of dimethyl sulfoxide. The enhancement of the fluorescence intensity of thrombin in the presence of dimethyl sulfoxide reflects the contribution of more exposed tryptophanyl residues. The alteration of the conformation of the enzyme structure due to the perturbation of the aqueous medium by dimethyl sulfoxide, has been attributed to these observed effects.

Long-Term Use of Minidose Heparin in Post-Myocardial Infarction

The value of heparin has been generally accepted in the prevention and treatment of venous thromboembolic phenomena. However, the use of heparin in the prevention of recurrent myocardial infarction (MI) in patients who have already experienced an MI is still being debated.

Identification of Individuals With Covert Ischemic Thrombotic Cerebrovascular Disease: A Discriminant Function Analysis

Sixty-one men with ischemic thrombotic cerebrovascular disease (ITCVD) and 61 age-matched controls (±three years) were studied to select risk factors which would identify ITCVD-prone individuals from a healthy population. Numerous clinical and biochemical measurements were made on all subjects including a three-hour oral glucose tolerance test concomitant with the immunoreactive insulin response (IRI). The following parameters were selected as risk factors for ITCVD from the many variables assessed: elevated systolic blood pressure; triglycerides; uric acid; glucose one-hour, two-hour and three-hour levels; and IRI two-hour and three-hour levels. There is also a higher prevalence of abnormal lipoproteins (mostly type IV), abnormal EKG, and hypertension in ITCVD. A stepwise linear discriminant function analysis was used to select the risk factors which best separate and classify the ITCVD and control subjects. These primary risk factors are the IRI three-hour level, systolic blood pressure, abnormal lipoproteins, and glucose two-hour level. Utilizing only these four primary risk factors in a discriminant function, 84.4% of the subjects were correctly classified. The contribution of the other variables is minimal in the discriminant function, but they are applicable as individual risk factors of ITCVD.

Biochemical analyses of human papillary muscles and guinea pig ventricles in failure.

Summary Studies were made on (a) human papillary muscles derived during open heart surgery in various degrees of failure, and (b) experimentally produced failing guinea pig hearts. There was a significant decrease in norepinephrine from 1.22 to 0.34 μg/g in the experimental guinea pig hearts as compared to normal. There was also a significant decrease in norepinephrine from 0.37 to 0.16 μg/g in human papillary muscle as the degree of failure increased. Glucose-6-phosphate decreased significantly from 0.30 to 0.09 μmoles/g in the human papillary muscles with increasing degree of failure. Similarly there was a significant rise in glycogen from 3.42 to 6.56 mg/g in the guinea pig hearts and from 5.69 to 8.89 mg/g in the papillary muscles. The differences in glycolysis and citric acid cycle intermediates were not significant. The ATP/ADP ratio decreased from 3.84 in grade II failure to 2.47 in grade IV failure. This ratio is a reflection of the degree of aerobiosis in mitochondria metabolism and indicates in failure a shift to more aerobiosis. The values on guinea pig heart adenyl cyclase activity were not statistically different, being 16.9 and 18.7 μmoles/100 g of tissue/15 min.