Polyacrylamide Gradient Gel Electrophoresis Research Articles

Insulin resistance is not a major determinant of low-density lipoprotein particle size

The relationship between low-density lipoprotein (LDL) peak particle diameter and insulin sensitivity, very—low-density lipoprotein (VLDL) + intermediate-density lipoprotein (LDL) triglyceride, cholesterol, and apoprotein B, postprandial lipemia, and LDL + high-density lipoprotein (HDL) triglyceride was assessed. The subjects were 101 healthy males aged 15 to 45 years. Sixty-one subjects (60.4%) were offspring of a parent with coronary artery disease before age 60, and 40 subjects (39.6%) had no parental history of coronary artery disease. LDL peak particle diameter was measured following polyacrylamide gradient gel electrophoresis. An insulin sensitivity index (S i) was determined from a frequently sampled intravenous glucose tolerance test using a minimal modeling method. A fat tolerance test was performed with a test meal containing 70 g/m 2 fat, with triglyceride concentrations measured hourly for 12 hours. LDL peak particle diameter was significantly correlated with body mass index (BMI) ( r = −.282, P < .01), waist to hip ratio ( r = −.291, P < .01), fasting triglyceride (logarithmically [log] transformed) ( r = −.556, P < .001), area under the postprandial triglyceride (log transformed) ( r = −.562, P < .001), VLDL + IDLL triglyceride (log transformed) ( r = −.321, P < .001), LDL + HDLL triglyceride (log transformed) ( r = .583, P < .001), and HDL cholesterol ( r = .347, P < .001), but there was no significant correlation with S i. Using stepwise regression analysis, LDL + HDL triglyceride showed the strongest relationship to LDL peak particle diameter, accounting for 34% of the variation in size. S I was not an independent predictor of LDL particle size. In conclusion, insulin sensitivity appears to have little influence on LDL particle size. The importance of LDL + HDL triglyceride should be ocnsidered a preliminary finding warranting varification in this and other populations.

Low-density lipoprotein particle size is not a discriminating marker for atherogenic risk in male offspring of parents with early coronary artery disease

The aim of this study was to assess the importance of low-density lipoprotein (LDL) particle size as a marker of atherogenic risk in male offspring of a parent with early coronary artery disease (CAD) before the age of 60 years. CAD-positive (CAD +) offspring were recruited into two groups based on age, 15 to 30 years (In = 20) and 31 to 45 years in = 41), and matched to CAD-negative (CAD −) offspring by age and body mass index (BMI) (n = 20 and 21 per group). LDL peak particle diameter was assessed by polyacrylamide gradient gel electrophoresis. There was no significant difference in LDL peak particle diameter between CAD + and CAD − offspring (26.2 ± 0.1 v 26.2 ± 0.1 nm, mean ± SE). There was also no difference between CAD + offspring and CAD offspring when comparisons were made within their own age group (26.5 ± 0.1 nm in younger CAD + offspring v 26.2 ± 0.1 nm in younger CAD − offspring, and 26.0 ± 0.1 nm in older CAD + offspring v 26.1 ± 0.2 nm in older CAD − offspring. Peak particle diameter was significantly greater in younger CAD + offspring than in older CAD + offspring (26.5 ± 0.1 v 26.0 ± 0.1 nm, P < .05). We conclude that small LDL particle size is not a discriminating marker for early atherogenic risk, and that measurement of LDL particle size has limited value in the assessment of coronary risk, at least in the age ranges we studied.

High density lipoprotein (HDL) particle composition in patients with end stage renal failure (ESRF) on chronic dialysis.

Hypertriglyceridaemia, low high density lipoprotein (HDL) cholesterol level and reduced LDL particle size are the major features of uraemic dyslipidaemia. They are also found in the Insulin Resistance Syndrome. To examine alterations in HDL composition in patients on chronic dialysis and their relationship with insulin resistance. HDL particle size was determined in 33 patients on chronic haemodialysis (HD), 27 on chronic ambulatory peritoneal dialysis (CAPD) and 32 control non-diabetic subjects (C) without renal disease by non-denaturing 3-30% polyacrylamide gradient gel electrophoresis. A weighted HDL particle size score was calculated taking into account both HDL particle size and percentage total HDL protein concentration of each HDL band of the individual. Lipid and apolipoliprotein concentrations were determined in HDL2 and HDL3 particles obtained by sequential ultracentrifugation. In a subset of 24 control subjects and 22 subjects on HD, insulin sensitivity was also determined by an intravenous glucose tolerance test (IVGTT). HDL particles were found to be more triglyceride enriched and apoAI depleted in subjects on HD even though plasma triglyceride level was highest in patients on CAPD. Five subpopulations of HDL particles were identified by gradient gel electrophoresis in all subjects combined. In the subgroup of subjects who underwent IVGTT, the weighted HDL particle size score correlated positively with HDL cholesterol level (r = 0.6, p < 0.0005), LDL particle size (r = 0.47, p < 0.001), and insulin sensitivity (r = 0.48, p < 0.001), and negatively with plasma triglyceride level (r = 0.37, p < 0.01). We conclude that even though HDL cholesterol is reduced to a similar level in subjects on both forms of dialysis for end stage renal failure, abnormalities of HDL composition are more marked in subjects on HD. Reduction in HDL particle size is linked with insulin resistance and accompanies reduction in LDL particle size and hypertriglyceridaemia.

Associations of age, adiposity, menopause, and alcohol intake with low-density lipoprotein subclasses.

We used nondenaturing polyacrylamide gradient gel electrophoresis to examine the associations of age, adiposity, menopause, and alcohol intake with LDL subclasses in 355 individuals. The absorbency of protein stain was used as an index of mass concentrations at intervals of 0.05 nm within seven LDL subclasses: LDL-IVB (22.0 to 23.2 nm), LDL-IVA (23.3 to 24.1 nm), LDL-IIIB (24.2 to 24.6 nm), LDL-IIIA (24.7 to 25.5 nm), LDL-II (25.5 to 26.4 nm), LDL-I (26.0 to 28.5 nm), and intermediate-size lipoproteins (ISL, 28.0 to 32.0 nm). Age and alcohol intake were obtained from questionnaires, and body mass index was computed from clinic measurements of weight and height. In adult men, body mass index correlated positively with LDL-III, and alcohol intake correlated positively with larger LDL-I. Age was positively correlated with LDL-IIIA and ISL in both men and women and with LDL-IIIB and LDL-II in women. Postmenopausal women had higher LDL-IIIA, LDL-II, and ISL than both premenopausal and premenarchal females. Adult males, > or = 18 years old, had higher levels of LDL-IIIA and LDL-II than younger males. Adjustment for fasting plasma triglyceride levels eliminated the significant associations between age and LDL-IIIA in both men and women and between age and LDL-II in women. Partial correlation analyses showed that reductions in the LDL peak diameter associated with increasing age, male sexual maturation, menopause, and adiposity are attributable to increases in the LDL-IIIA subclass. Thus, densitometric measurements of protein-stained gradient gels reveal specific relationships between LDL subclasses and age, adiposity, and alcohol intake beyond those identified by the LDL peak or average diameter.

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Changes in nutrient and protein composition of cat milk during lactation

Abstract Objective To evaluate changes in the nutrient and protein composition of cat milk during lactation. Animals 12 lactating domestic shorthair cats. Procedure Milk samples collected on days 1, 3, 7, 14, 28, and 42 after parturition were analyzed for concentrations of nitrogen, nonprotein nitrogen, casein, whey proteins, amino acids, total lipids, lactose, citrate, minerals, and trace elements. Individual milk proteins (caseins and whey proteins) were analyzed by use of polyacrylamide gradient gel electrophoresis. Results True protein concentration ranged from 6.3 to 8.6% and was as high in mature milk as in colostrum. Nonprotein nitrogen as a portion of total N was constant (approx 8%), as was the whey-to-casein ratio (approx 50:50). Total lipid concentration was high (9.3%) in colostrum, rapidly decreased, then increased to 9% in mature milk. Lactose concentration was constant at 4%. Milk calcium, iron, and copper concentrations increased markedly during lactation, and magnesium and zinc values remained constant. Colostrum and early milk had a low Ca-to-P ratio of 0.4:0.9. Although calcium concentration increased with time, phosphate concentration also increased so that the Ca-to-P ratio remained constant in mature milk at 1.0: 1.2. The major whey proteins had molecular weights of approximately 14,000, 19,000, 40,000 and 80,000. The 80,000 protein (possibly lactoferrin) decreased in concentration during lactation. Two major casein subunits of approximately 28,000 and 33,000 were found, and both increased during early lactation. Conclusions Nutrient composition of cat milk and, thus, provision of nutrients to nursing kittens changes over time. (Am J Vet Res 1997;58:370-375)

High-density lipoproteins from probucol-treated patients have increased capacity to promote cholesterol efflux from mouse peritoneal macrophages loaded with acetylated low-density lipoproteins.

To elucidate the anti-atherogenic effect of probucol, high-density lipoprotein (HDL) was isolated from probucol-treated patients (n = 14) and compared with that from control subjects (n = 12). The HDL obtained from probucol-treated patients was low in cholesteryl ester (CE) in comparison with that from control subjects (21.3 +/- 3.9 mol per cent vs. 27.6 +/- 3.2 mol% of total lipids. P < 0.001), and the peak diameters of patients' HDL were significantly smaller than those of control subjects on 4-30% non-denaturing polyacrylamide gradient gel electrophoresis (10.6 +/- 0.6 nm vs. 12.1 +/- 0.4 nm, P < 0.001). These data may be explained by the increased cholesteryl ester transfer protein (CETP) activities of probucol-treated patients (129 +/- 12% of control subjects, P < 0.001). The in vitro ability of HDL to remove CE from lipid-laden macrophages induced by incubation with acetylated low-density lipoprotein (Ac-LDL) was studied. The small and CE-poor HDL obtained from probucol-treated patients had a greater capacity to promote CE efflux from macrophages than did control HDL (59.8 +/- 6.9% vs. 44.2 +/- 5.4%, P < 0.01). Furthermore, the ability of HDL to promote cholesterol efflux correlated negatively with the CE content and particle diameter of HDL (r = -0.561 and r = -0.583 respectively; P < 0.01). When the inhibitory effect of HDL on the incorporation of [14C]-oleate into cellular cholesteryl ester was compared, the HDL from patients and control subjects inhibited CE formation to a similar extent. The enhanced ability of probucol-treated patients' HDL may, therefore, be involved in the acceleration of hydrolysis of the CE pool in macrophages. Taken together, we conclude that CETP plays a crucial role in making HDL more active in its anti-atherogenic function by reducing CE and making HDL smaller, and that probucol may enhance reverse cholesterol transport by activating CE transfer in vivo. The current study demonstrated, for the first time, that HDL modified by enhanced CETP activity in vivo is potentially anti-atherogenic.

Specific reduction of plasma large, light low-density lipoprotein by a bile acid sequestering resin, cholebine (MCI-196) in type II hyperlipoproteinemia

The effect of a bile acid sequestrant, cholebine (3 g/day), on plasma lipoprotein subfractions was investigated in 16 patients with type II hyperlipoproteinemia. Activities of low density lipoprotein (LDL)-receptor and activities of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) were assayed to address the mechanism of cholebine-induced changes in plasma lipoprotein subfractions. Twelve weeks of treatment with cholebine reduced plasma levels of total cholesterol (TC) and LDL-cholesterol (C) by 8.3±8.1% (mean±S.D.) and 14.4±11.9%, respectively ( P<0.001), but did not affect plasma levels of high density lipoprotein (HDL)-C. Cholebine significantly reduced plasma levels of LDL 1-C (1.019< d<1.045) by 22.9±18.9% ( P<0.001) but did not affect plasma levels of very low density lipoprotein (VLDL)-C, intermediate density lipoprotein (IDL)-C, LDL 2-C (1.045< d<1.063), HDL 2-C, and HDL 3-C ( d>1.125). Gradient polyacrylamide gel electrophoresis (PAGE) revealed that cholebine reduced large LDL in plasma but had almost no effects on small LDL and HDL subfractions. Cholebine did not alter the activities of LCAT and CETP. LDL-receptor activities of cultured lymphocytes negatively correlated with the reduction in plasma levels of LDL-C ( r=−0.500, P<0.05), IDL-C ( r=−0.581, P<0.02), and LDL 1-C ( r=−0.610, P<0.01), respectively. Thus, cholebine seems to reduce further the plasma levels of IDL and large, light LDL in patients with lower LDL-receptor activities. We conclude that cholebine only reduces plasma levels of large, light LDL. This may be due to the stimulation of hepatic LDL-receptor activity.

Purification and Characterization of Hepatic Aldehyde Oxidase in Male and Female Mice

To examine whether the hepatic aldehyde oxidases of male and female mice, which exhibit sex-related differences in benzaldehyde oxidation, are qualitatively different, we purified the enzymes from untreated male and femaleddymice and testosterone-pretreated female mice by sequential chromatography of benzamidine–Sepharose 6B and DEAE-5PW columns and characterized the enzymes. Purified enzymes from untreated male and female mice and from testosterone-treated females gave a single protein band atMr265K andMr138K on native and SDS–polyacrylamide gradient gel electrophoresis, respectively. The susceptibilities of the enzymes from both sexes to inhibitors such as menadione, norharman, quinacrine, estradiol, and SKF 525-A were also indistinguishable. However, theKmvalues for benzaldehyde,p-dimethylaminocinnamaldehyde, and 2-hydroxypyrimidine were two- to fourfold higher in the untreated female enzyme than in the untreated male enzyme, while the testosterone-induced female enzyme showedKmvalues similar to those of the male enzyme. These results indicate that the hepatic aldehyde oxidases of the sexes are quite similar with respect to molecular size and susceptibility to inhibitors, but their kinetic properties are somewhat different, suggesting the existence of microheterogeneity in sex differences. It also suggests that treatment of female mice with testosterone might induce the male-type enzyme.

Biosynthesis of Teichuronic Acid in the Bacterial Cell Wall: PURIFICATION AND CHARACTERIZATION OF THE GLUCOSYLTRANSFERASE OF MICROCOCCUS LUTEUS

This report describes what is, to our knowledge, the first purification to near homogeneity of an enzyme involved in the biosynthesis of the teichuronic acid of Micrococcus luteus cell walls. The glucosyltransferase of M. luteus, which participates in the biosynthesis of teichuronic acid, was solubilized from cytoplasmic membrane fragments by extraction with buffer solutions containing the detergents Thesit (dodecyl alcohol polyoxyethylene ether; 1 mg/ml) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (0.5 mg/ml). The detergent-solubilized enzyme was purified 150-fold, with a recovery of 13% by adsorbent column chromatography, ion-exchange chromatography, gel filtration, and preparative nondenaturing gradient polyacrylamide gel electrophoresis. On the basis of its mobility on native gradient gel, the glucosyltransferase was estimated to have a molecular mass of 440 kDa. The purified native enzyme was a multisubunit protein consisting of subunits of two sizes; their molecular masses were determined to be 52.5 and 54 kDa, respectively, by observation of the mobility of the protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was approximately 5.

Measurement of small high density lipoprotein subclass by an improved immunoblotting technique

An improved method of immunoblotting of plasma onto agarose gel matrix containing antiapolipoprotein A-I is described. Fresh plasma samples were subjected to gradient polyacrylamide gel electrophoresis (4-25%) followed by electrotransfer onto agarose gel layer containing antiapolipoprotein A-I. This method was compared with immunoblotting onto nitrocellulose where the transfer onto agarose gel matrix has been shown to be more convenient, quantitative, and can be kept permanently. Plasma apolipoprotein A-I was found to be distributed among regions of varying molecular weights ranging from 43,000 to 800,000. A small size fraction of molecular weight range of 43,000-50,000 (small HDL) was found in normolipidemic and hyperlipidemic subjects. The proportion of the latter fraction varied considerably among subjects (range: 0.0-32%), being lower in normolipidemic subjects (mean +/- SEM: 11.6 +/- 1.4%), and higher in hyperlipidemic subjects (mean +/- SEM: 23.7 %/- 1.7%, P < 0.001). Physiological increase in the level of the small HDL was observed in normolipidemic subjects 4 h after fat ingestion (difference: 5.0%, P < 0.001); moreover, the level was higher in normolipidemic subjects who consumed moderate amounts of alcohol (mean +/- SEM: 17.9 +/- 1.2%, P < 0.001) compared with normolipidemic subjects who do not drink alcohol at all.

Detection of heparin-like glycosaminoglycans in normal human plasma by polyacrylamide-gel electrophoresis

This paper describes a procedure that allows detection of endogenous heparin present in normal human plasma by polyacrylamide-gel electrophoresis (PAGE). Plasma was submitted to proteolysis: an antithrombin III-dependent and heparinase I-sensitive anticoagulant activity was demonstrated in the supernatant of the digest. The supernatant was submitted to sequential fractionation with increasing concentrations of ethanol (25%, 50%, 60% and 65%, by vol.). Fractions were analyzed by PAGE for both glycosaminoglycan (GAG) and protein content. GAGs were detected by gradient PAGE (24–30%). The fraction obtained by 60% ethanol precipitation contained heparinase I-sensitive GAG. We show that GAGs co-precipitate with proteins. The SDS-PAGE of the material resulting from proteolytic digestion and subsequent ethanol fractionation, revealed three major bands. These peptides co-precipitated with plasma GAGs, mainly with the fraction obtained by 60% ethanol. We discuss the possibility that circulating endogenous heparin interacts with such peptides.

Atherogenic Modified LDL in Diabetes

This paper summarizes the recent findings on LDL atherogenic modifications in diabetic patients. LDL from diabetic patients, unlike LDL from healthy subjects, caused a significant increase in cholesterol content of cells cultured from unaffected human aortic intima, i.e., produced a direct atherogenic effect. LDL was divided into two fractions (nonbound and bound) by affinity chromatography on Ricinus communis agglutinin-agarose. The amount of bound LDL was significantly higher in diabetic patients compared with healthy subjects. Bound LDL was characterized by significantly lowered sialic acid content and a significantly increased fructosyl lysine level compared with unbound LDL, i.e., was desialylated and nonenzymatically glycosylated lipoprotein. The bound (desialylated), but not unbound (sialylated), LDL subfraction induced cholesterol accumulation in cultured cells. Bound LDL was also characterized by a significantly lowered content of neutral lipids and demonstrated increased electrophoretic mobility on agarose gel electrophoresis. Bound and nonbound LDL differed significantly in hydrated density and particle size, as was determined by density gradient ultracentrifugation and native polyacrylamide gradient gel electrophoresis. The results of this study have shown that the in vivo modified atherogenic LDL subfraction in the blood of diabetic patients is represented by small, dense, more electronegative, desialylated, and glycated LDL.

High prevalence of small LDL particles in non-insulin-dependent diabetic patients with nephropathy

To determine whether small-sized low density lipoprotein (LDL) is associated with a high incidence of coronary heart disease in diabetic nephropathy, we measured the LDL particle size in non-insulin-dependent diabetes mellitus (NIDDM) patients with various degrees of albuminuria ( n = 95) and age-, weight-matched non-diabetic control subjects ( n = 31). The diabetic subjects were divided into three groups, normoalbuminuric, microalbuminuric and macroalbuminuric NIDDM, based on the amount of albuminuria. The average diameter of LDL particles was determined by non-denaturing polyacrylamide gradient (2–16%) gel electrophoresis. The plasma lipid and lipoprotein concentrations were comparable between the non-diabetic controls and normoalbuminuric NIDDM, whereas the plasma triglyceride, very-low-density lipoprotein (VLDL) or LDL concentration was significantly increased in diabetic nephropathy. The mean LDL particle size was significantly smaller in microalbuminuric NIDDM compared with the controls or normoalbuminuric NIDDM, and the LDL size was further decreased in macroalbuminuric NIDDM. The incidence of small LDL (diameter < 255 Å) was remarkably increased in microalbuminuric (58%) and macroalbuminuric NIDDM (67%) compared to the control (13%) and normoalbuminuric NIDDM (27%). Corresponding to the decreased LDL size, the cholesterol content of the LDL was significantly depleted in NIDDM with nephropathy. The high prevalence of small LDL in diabetic nephropathy was also observed even when hypertriglyceridemic or hypertensive subjects were excluded from each group. The increment in triglyceride-rich lipoprotein ( d < 1.006) after oral fat-loading was increased, and postheparin lipoprotein lipase activity was decreased significantly in diabetic nephropathy. These abnormalities were significantly associated with LDL particle size. Multivariate regression analysis revealed that the amount of albuminuria was closely associated with the average LDL particle size, and this association was independent of the plasma triglyceride level. Neither insulin resistance nor glycemic control was directly associated with LDL particle diameter. The present study indicates that LDL particles become smaller in diabetic nephropathy, and this may be associated primarily with abnormal triglyceride metabolism. However, in addition to hypertriglyceridemia, other metabolic abnormalities caused by diabetic nephropathy may also be involved in the pathogenesis of small LDL particles.

Phospholipid transfer protein mediated conversion of high density lipoproteins generates pre β1-HDL

High density lipoproteins (HDL) subclasses can be differentiated by two-dimensional non-denaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) and subsequent immunoblotting. The quantitatively minor HDL-subclasses pre β 1-LpA-I and γ-LpE are initial acceptors of cell-derived cholesterol into the plasma compartment. In this study we analysed the effect of phospholipid transfer protein (PLTP) on the electrophoretic distribution of HDL-subclasses in plasma as well as the ability of plasma, pre β 1-LpA-I, and γ-LpE to take up [ 3H]cholesterol from labeled fibroblasts. Pre β 1-LpA-I but not γ-LpE disappeared during a 16 hours incubation in the absence of PLTP. During a one minute incubation pre β 1-LpA-I of pre-incubated plasma released 75% less [ 3H]cholesterol from radiolabeled fibroblasts than pre β 1-LpA-I of control plasma. Pre-incubation of plasma reduced the uptake of [ 3H]cholesterol by γ-LpE by 40%. Totally, the cholesterol efflux capacity of plasma decreased by 10% compared to the original sample. The amount of immunodetectable pre β 1-LpA-I increased when plasma was incubated in the presence of PLTP while the amount of immunodetectable γ-LpE did not change. After one minute incubation of PLTP-conditioned plasma with [ 3H]cholesterol-labeled fibroblasts, the amount of radioactive cholesterol taken up by pre β 1-LpA-I was twice as high as in control plasma whereas the amount of [ 3H]cholesterol taken up by γ-LpE remained unchanged. As a net result, treatment with PLTP increased the cholesterol efflux into total plasma by 40%. Together with results of previous studies our data suggest that the conversion of α-LpA-I 3 into α-LpA-I 2 by PLTP generates pre β 1-LpA-I but not γ-LpE. PLTP helps to enhance the uptake of cell-derived cholesterol by pre β 1-LpA-1 and, thereby, the cholesterol efflux capacity of normal plasma.

Evaluation of patterns of urinary proteins by SDS-PAGE in rats of different ages

The patterns of urinary proteins in rats of different ages were examined on SDS gradient polyacrylamide gel electrophoresis coupled with silver staining. Proteins were fractionated into at least 26 bands. Densitometric measurements were used to characterize protein excretion patterns. The results showed that proteinuria in newborn, young and adult rats is predominantly tubular, consisting of low molecular-weight species. Conversely, late adults and old rats had a mixed glomerular pattern, with a steadily increasing excretion of albumin, IgG and transferrin, as was the case of other high molecular-weight proteins. Fragments of both immunoglobulins and albumin were found in all urine samples assayed. In 1 month old rats the percentage of Tamm-Hörsfall (T-H) protein was higher ( P<0.01) than in the remaining groups studied. In newborns, relatively high albumin, IgG and transferrin percentages were detected, as well as an α 1-acid glycoprotein and carbonic anhydrase excretion ( P < 0.05 and P < 0.01 respectively) higher than that observed in the other age groups studied.

Evaluation of Common Electrophoretic Methods in Determining the Molecular Weight of Apolipoprotein(a) Polymorphs

Five different gel systems were evaluated for their utility in determining the molecular weights of apolipoprotein(a) (apo(a)) polymorphs by SDS polyacrylamide or agarose gel electrophoresis. Three linear polyacrylamide gradient gels (2–16% from Isolab (Akron, OH), 4–15% from Pharmacia (Piscataway, NJ), and 2.5–6% homemade), a 4% polyacrylamide, and a 1.5% agarose gel were examined. Crosslinked phosphorylase B oligomers served as molecular weight standards. Molecular weights of four different apo(a) polymorphs were determined in each gel system and compared to values measured previously by sedimentation equilibrium. The results indicate that molecular weights obtained by gradient polyacrylamide gel electrophoresis were within 10% and often not statistically different from values acquired by sedimentation equilibrium. The use of homogenous 4% polyacrylamide and 1.5% agarose gels led to molecular weights that were overestimated by 20 and 60–70%, respectively. ApoB100, which is a commonly used molecular weight marker, was found to have anomalously fast mobility in each of the four polyacrylamide gel systems. Because its use would lead to overestimated apo(a) molecular weights, it was not useful as a molecular weight standard. Our results indicate that SDS–gradient polyacrylamide gel electrophoresis with cross-linked phosphorylase B as standard is a suitable gel system for evaluating apo(a) molecular weights.

Polyacrylamide gel electrophoretic serum protein patterns of acute inflammation induced by intramuscular injection of turpentine in young broiler chickens.

Changes in the serum protein patterns accompanying inflammation in young broilers were examined by polyacrylamide gradient gel electrophoresis. Sera were obtained from blood of 4-week-old male broilers after induction of acute inflammation by an injection of 0.5 ml/kg turpentine. Electrophoretic patterns at 48 hr after injection, showed an increase in transferrin (Tf) and a segment containing albumin (Alb) with multiple peaks. The unbound iron binding capacity (UIBC) increased by 10 times, and the serum iron (SI) concentration in acute inflammation was reduced to one-half its initial value. These are considered to be typical changes in serum proteins and iron after acute inflammation.

Accelerated Stability Studies of Heparin

The objective of this study was to extend our understanding of the stability of heparin. Sodium heparin, derived from porcine intestinal mucosa, was first incubated in 0.1 N hydrochloric acid and 0.1 N sodium hydroxide at 30 and 60 °C and sampled at times ranging from 0 to 1000 h. The absorbance spectra of the products formed under basic conditions showed an ultraviolet maxima at 232 nm associated with chemically catalyzed β-elimination at the uronic acid residues. The products formed under acidic conditions showed a decreased staining intensity consistent with desulfation and a decrease in molecular weight corresponding to hydrolysis of glycosidic linkages when analyzed by gradient polyacrylamide gel electrophoresis. Heparin samples were next prepared in 10 mM sodium phosphate buffer at pH 7.0 in sealed ampules that had been flushed with nitrogen and incubated at 100 °C. Samples taken at times ranging from 0 to 4000 h were then analyzed. Heparin was relatively stable over the first 500 h, after which it rapidly degraded. Heparin, assayed using both anti-factor Xa and anti-factor lla amidolytic methods retained 80-90% of its activity over the first 500 h, but these activities dropped precipitously, to ~6% and ~0.5% of the initial activity at 1000 h and 2000 h, respectively. This rapid decomposition began only after the buffering capacity of the solution was overwhelmed by acidic degradants, which caused the pH to decrease. Decomposition processes observed under these conditions included the endolytic hydrolysis of glycosidic linkages and loss of sulfation, particularly N-sulfate groups, and were similar to the degradation processes observed in 0.1 N hydrochloric acid. This study provides initial observations on heparin degradation pathways. More complete, quantitative studies and studies leading to the isolation and characterization of specific degradants are still required.

HDL subfractions distribution in renal transplant recipients: lack of evidence of a reduction of HDL2 particles.

Since the high rate of cardiovascular disease in renal transplant recipients, alterations of lipoprotein profile in such patients were extensively evaluated, but the HDL subclass profile was not completely clarified. Renal transplant recipients usually show normal to high plasma levels of HDL cholesterol, even if some investigations suggested a persistence of low HDL2 levels: this was not useful in terms of cardiovascular protection. We designed this study in order to evaluate HDL subfractions distribution in renal transplant recipients. We studied 55 renal transplant recipients, treated with prednisone, azathioprine and/or cyclosporine, and 34 healthy normolipidemics as controls. In all subjects cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, apolipoproteins A-I and B levels and HDL subfractions, as determined by nondenaturing polyacrylamide gradient gel electrophoresis, were assayed. Renal transplant recipients had cholesterol, triglycerides, LDL cholesterol and apolipoproteins A-I and B levels significantly higher than controls; HDL cholesterol levels were slightly, but not significantly, increased in comparison with controls (respectively 51.1 +/- 15.5 and 46.1 +/- 10.8 mg/dl). Multivariate analysis showed that only the time since transplantation was significantly associated with HDL cholesterol levels. When HDL subfractions were analyzed, renal transplant recipients presented significantly lower levels of HDL3a and HDL3b and, in males, higher levels of HDL2b than controls. HDL cholesterol levels were positively correlated with HDL2b levels in both renal transplant recipients and controls, and negatively correlated with HDL3b in controls. In conclusion, in renal transplant recipients, we failed to demonstrate any decrease of HDL2 particles. On the basis of a nonatherogenic HDL profile, we suggest that the increased cardiovascular risk in renal transplant recipients might be accounted for by other risk factors.