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In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18-36 years) and eighteen individuals without diabetes (17-35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (microg/l) and CEC (microg/l) were significantly higher in DM (55.73 +/- 29.43, 521.47 +/- 239.13, respectively) compared to controls (24.25 +/- 10.26, 262.85 +/- 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.

To assess the relationship between heparin and the associated increase in nonesterified fatty acids (NEFA) and their possible influence on Na,K-ATPase during hemodialysis, we studied two groups of patients: (1) 12 patients on chronic hemodialysis dialysed with heparin and (2) 6 patients dialysed without heparin. All 12 patients who received heparin anticoagulation had a 7-fold rise in NEFA on average and also had an increase in circulating inhibitors of Na,K-ATPase assayed by 3H-ouabain displacement from Na,K-ATPase and/or by effect of plasma on the uptake of 86Rb by rat aortic rings. Serial assays in 3 of the patients receiving heparin showed NEFA and inhibitory changes to be at or near maximum within 30-60 min. Of the individual NEFA, the greatest relative increases were in oleic (18:1) and linoleic (18:2) acids, and the strongest correlations were between linoleic acid and both 3H-ouabain displacement (r = 0.94) and 86Rb uptake (r = 0.86). However, a small and slower increase in NEFA also occurred in 3 of the patients dialysed without heparin. We conclude that heparin anticoagulation during dialysis leads to a rapid and marked increase in circulating NEFA, particularly the unsaturated fatty acids, with a corresponding interference with Na,K-ATPase activity. The clinical significance of these findings is unknown. The rise in NEFA during dialysis without heparin in some patients suggests that factors other than heparin may also contribute to the rise in NEFA.

Lipid composition of the myocardium and in vitro lipid metabolism were studied in hearts from young rats after 30 days of treatment with triiodothyronine (100 microgram/kg per day) and in heterotopically isotransplanted hearts of inbred adult rats 6 days after surgery. The former served as an experimental model of cardiac hyperfunction, while the latter, empty beating hearts, served as a model of cardiac hypofunction. In hearts from hyperthyroid animals the concentration of phosphatidylcholine, phosphatidylethanolamine, cardiolipin, and the incorporation of 14C-labelled palmitic and erucic acid into these phospholipids were increased significantly as compared with controls. In contrast, the triglyceride concentration and the incorporation of palmitate into triglyceride was significantly decreased. In transplanted hearts, the phospholipid concentration and the incorporation of 14C-labelled fatty acids into phospholipids were significantly decreased as compared with the hearts of the inbred host rats of the same age. The results indicate that the mechanical performance of the heart affects the phospholipid composition, which may be a reflection of increased or decreased proliferation of subcellular membranes in sustained cardiac hyper- or hypo-function.

To elucidate the effects of chronic alcohol ingestion in monkeys a synthetic, adequately balanced, fluid diet providing 40% of total calories from ethanol was gavaged through a stomach tube daily over a period of three months. Clinical, biochemical, radioisotope, and histopathological studies were performed at the beginning and end of the experiment. It was observed that chronic alcohol feeding at this dose level caused maked accumulation of triglycerides, cholesterol, and phospholipids in the serum and the liver. In the heart triglycerides and cholesterol ester were increased. Incorporation studies showed increased synthesis of triglycerides in the heart muscle and liver. Histologically the heart showed fatty change of the myocardium and evidence of focal myocytolysis, atrophy of muscle bundles, and early fibrosis. The liver showed generalized fatty change but no cirrhosis.

This method for assaying digoxin in serum with improved specificity combines small-column extraction of serum, "high-performance" liquid chromatography, and RIA of the eluted fractions. Analytical recoveries of 1.0, 0.5, and 0.1 microgram/L standards were 95%, 93%, and 84%, respectively. The CVs for duplicates and replicates of sera with values of 0.5 to 1 microgram/L were 4 to 6%. Fifty-nine sera from 50 patients receiving digoxin were so studied. All digoxin metabolites appear to cross react with antibody to digoxin to various degrees. The most polar metabolites were quantitatively the most important, their average cross reactivity being 33%. For eight patients the value for digoxin by the present method was less than 60% of the RIA value. Sera from nine patients not taking digoxin but with falsely high digoxin values were also studied by the present method. The digoxin peak was well resolved from those for (a) digoxin metabolites (except dihydrodigoxin), (b) digitalis-like factors in neonates and in patients with renal failure or combined hepatic and renal failure, and (c) two cross reacting drugs and their metabolites.

Sarcolemmal and mitochondrial phospholipids were extracted from normally perfused and ischemic regions of the dog heart and the composition of these extracts was analyzed. Relatively pure sarcolemmal fraction obtained from myocardium subjected to 3 h of ischemia exhibited a significantly lower concentration of phospholipids than that obtained from normal myocardium. In particular, phosphatidylcholine and phosphatidylethanolamine were reduced by approximately 33%. The fatty acid content in the sarcolemmal phospholipid fraction was also reduced by approximately 30% without any change in the relative composition. In the mitochondrial fraction, the relative phospholipid composition was also altered by ischemia; the major components (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) being reduced by approximately 15-20%. An attempt was made to correlate these biochemical changes with ultrastructural lesions observed electron microscopically. These observations revealed extensive regional variability and a wide heterogeneity in the extent of ultrastructural damage evident in the different organelles even in a single cell. This may suggest that ischemic damage, in the early stages, may advance at widely varying rates in different regions. Our findings demonstrate that significant biochemical and structural disorganization occurs during 3 h of ischemia in the myocardium and raise the possibility that one of the initiating events is the activation of sarcolemmal and mitochondrial phospholipases.

Stereospecific degradation and combined gas chromatographic--mass spectrometric (gc/ms) analysis were employed in a detailed investigation of the triacylglycerol structure of mustard seed oil and of the triacylglycerols transiently accumulating in the hearts of young rats receiving the oil in their diet. It was shown that feeding of mustard seed oil at 40% of the daily caloric requirement resulted in a deposition of cardiac triacylglycerols containing a high proportion of enantiomers of a positional distribution and molecular association of fatty acids which were closely similar to those found in the dietary oil. Complete structures were derived for a total of 88 species representing 75 to 85% of the triacylglycerols. About 90% of the accumulated triacylglycerol contained at least one long-chain (C20--C22) monounsaturated fatty acid per molecule. The long-chain acids were confined mainly to the primary positions and preferentially to the sn-3-position of the glycerol molecule. The dietary lipidosis, is, therefore, accompanied by little or no accumulation of the normal rat tissue triacylglycerols containing C16 and C18 fatty acids. It is suggested that the deposition and eventual clearance of the enantiomeric long-chain triacylglycerols in the rat heart during mustard seed oil feeding may be largely a result of a gradual change in specificity of the cardiac lipases.

Serum digoxin and metabolites were assayed in plasma and urine by HPLC in 10 dialysis-dependent patients with end-stage renal failure (group I) and in five patients with comparatively normal renal function (group II) after ingestion of 150 muCi 3H-digoxin-12 alpha. Thirteen patients were on maintenance digoxin therapy and were at steady state. Metabolites found regularly but usually in small amounts, were 3 beta-digoxigenin and its mono- and bis-digitoxosides, and 3-keto and 3 alpha(epi)-digoxigenin. Quantitatively the most abundant metabolites were polar and averaged 26% (7 to 76) of the radioactivity in plasma 6 hr after drug, and 60% (11 to 88) for digoxin for all 15 patients. Neither values between group I and II for the polar metabolites nor digoxin differed significantly. The metabolites reacted with antibody to digoxin to varying degrees and may make up an important component of the serum digoxin concentration when determined by standard radioimmunoassay. In some patients, digoxin undergoes extensive biotransformation, mainly, we suggest by hydrolysis, oxidation, epimerization, and conjugation to polar end-metabolites.

In the present work the uptake and utilization of [14C]erucic acid by the perfused rat heart has been investigated and compared with those of [14C]-palmitic acid. Both fatty acids were found to be taken up by the heart at the same rate. On the other hand, the incorporation of erucic acid into tissue lipid during 30 min perfusion were significantly high and CO2 production low as compared with palmitic acid. Incorporation of erucic acid into diacylglycerol, triacylglycerol and cholesterol ester was considerably higher than that of palmitic acid. During a 30-min period, a large amount of [14C]erucic acid was accumulated in tissue fatty acid fraction. Similarly, relatively high labelling was found in the fatty acid and diacylglycerol fraction during the initial 300 s of perfusion with erucic acid. When [14C]erucic acid and unlabelled palmitic acid was used, the radioactivity was very high in the fatty acid fraction of the heart lipid in comparison with the experiment when [14C]palmitate and unlabelled erucic acid was used. Therefore, erucic acid is poorly oxidized by the heart and is preferentially incorporated into heart lipids. There was relatively high incorporation of [14C]erucic acid into diacylglycerol and addition of unlabelled palmitic acid tended to decrease it, probably converting more diacylglycerol to triacylglycerol. When [14C]palmitic acid and erucic acid were used together, incorporation to triacylglycerol was high and diacylglycerol low. These results, therefore suggest that palmitic acid is a more suitable acyl donor than erucic acid for the C-3 position of triacylglycerol, especially when the diacylglycerol contains erucoyl moieties.

We assayed plasma Na,K-ATPase inhibitory activity due to total lipids and lipid fractions. The effect of dialysis on the Na,K-ATPase inhibitory activity was also studied. Plasma lipid extracts from 11 healthy volunteers and 9 dialysis-dependent patients (pre and post dialysis) were separated into neutral lipids and phospholipids. Further fractionation was by thin layer chromatography. These lipid fractions were analyzed for Na,K-ATPase inhibitory activity by displacement of [3H]-ouabain from hog brain Na,K-ATPase. Total inhibitory activity was significantly increased (p less than 0.001) in the post-dialysis plasma compared to pre-dialysis plasma of the same patient group and to controls (482, 85 and 78 nmol/L respectively; means of the groups in digoxin equivalents). The major inhibitory activity was associated with non-esterified fatty acids with modest contributions from four other lipid fractions. Our results show that endogenous lipids are major plasma Na,K-ATPase inhibitors in vitro under these assay conditions.

Phospholipid composition of mitochondrial and lysosomal fractions of the rabbit heart under hypoxic conditions was studied. Only when the two fractions were incubated together under hypoxic conditions for 10 or 60 minutes, did phosphatidylethanolamine and phosphatidylcholine concentrations of mitochondria and lysosomes decrease significantly, suggesting that there is certain interaction between these organelles under induced hypoxia in vitro.

We have studied the incorporation of various long chain fatty acids into tissue lipids using various heart preparations. We found that individual fatty acids are incorporated into various lipid fractions at unequal rates. In particular, erucic acid was shown to be a poor acyl donor for various acylation reactions. Labeling of triacylglycerol by exogenous fatty acid was found to be extremely rapid in the perfused rat heart and in the in vivo heart.

Rats of weaning age were feed for a period of 1 or 3 weeks either a low fat diet (laboratory stock diet) or a semi-synthetic diet containing 20% by weight of either corn oil (2/3 of the total fatty acids consisted of linoleic acid) or mustard seed oil (1/3 of the total fatty acids were comprised of erucic acid). Feeding of a diet rich in erucic acid for 1 week increased by twofold the cholesterol ester content of the aorta. However, the concentrations of unesterified cholesterol and individual phospholipids in the aorta were uninfluenced by hyperlipemia induced by this diet. Fatty acid analyses indicated that erucic (22:1) and eicosenoic (20:1) acids are found in the triglyceride and cholesterol esters of the aorta in the proportion similar to that found in the plasma triglyceride and cholesterol esters. Our results demonstrated further that a significantly greater quantity of 14C-labelled erucate than labelled palmitate, oleate or linoleate was incorporated into the cholesterol ester fraction of the control aorta. The incorporation rates of erucate and linoleate into phosphatidic acid were more rapid, while the rates of erucate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine were significantly slower than those of other fatty acids.

Digitalis-like factors were assayed by radioimmunoassay of digoxin in 6 bile samples obtained from patients at autopsy and in plasma from three patients with combined hepatic and acute renal failure. None of the patients received digoxin. Digitalis like factor values in bile samples were 23 to 85 nmol digoxin equivalents/1. Bile salt concentrations ranged from 38-104 mmol/l in the bile and 28-184 mumol/l in the plasma of these subjects. Bile, plasma digitalis like factor extracts and bile salt standards (0.1-3 mM) showed concentration dependent displacement of [125I]-digoxin from digoxin antibody, inhibition of hog brain Na,K-ATPase and displacement of [3H]-ouabain from Na,K-ATPase. The concentration-displacement curves suggest that bile salts could account for 50-79% of the total digitalis like factors in the six bile samples and 2-7% in the plasma of the three patients. High performance liquid chromatographic fractionation of a bile sample showed digitalis like factor peaks co-eluating with standards of tauro- and glycocholate, tauro- and glycochenodeoxycholate and tauro- and glycodeoxycholate. These bile salt peaks accounted for 78% of the total digitalis like factors in all high performance liquid chromatographic peaks in bile, but only 7% of the total digitalis like factor activity in all high performance liquid chromatographic peaks in an extract of plasma from one of the patients with hepatic and renal failure. The bile salts appear to be examples of endogenous digitalis like compounds which do not act by simple competitive ligand binding to antidigoxin antibody and Na,K-ATPase. They make an important contribution to digitalis like factor activity in bile, but not in plasma.

When male weaning rats were fed a high fat-high erucic acid and diet (mustard seed oil) for 1 week, there were increases in the triglyceride and erucic acid contents in the myocardium, and an increase in the rate of incorporation of [1-14C]palmitic acid into triglyceride in the perfused heart, in comparison to rats that had consumed a high fat-high linoleic acid diet (corn oil) or a low fat diet (laboratory chow). However, continuation of the mustard seed oil diet (3-6 weeks) tended to normalize these biochemical parameters. The size of the papillary muscle isolated from the mustard seed oil-fed rats was greater than that of the muscle of the chow-fed rats, and its developed isometric tension was lower. Electron microscopic examination revealed that, in the hearts of rats having eaten the mustard seed oil diet, there were isolated foci where the number and size of mitochondria appeared to be increased.