The interaction of pyridoxal phosphate with aspartate apoaminotransferase.

The rate of biniding of pyridoxal phosphate to the apoenzyme of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was measured by adsorption spectroscopy and by formation of active enzyme. At pH 5.1 and 8.3 the binding of coenzyme follows saturation kinetics. The binding process thus involves at least two steps. The rate of pyridoxal phosphate binding to the apoenzyme is dependent on the anion present in the pH 8.3 triethanolamine buffer. Chloride activates somewhat at very low concentrations. Phosphate and its methyl, ethyl, and phenyl esters are very effective inhibitors of the recombination in that 0.2--0.4 mM inhibit the rate of coenzyme binding by 50%. This is below the physiological concentration of phosphate. Sulfate also inhibits the rate of binding, but nitrate and acetate have little effect.

Identification of Lys190 as the primary binding site for pyridoxal 5'-phosphate in human serum albumin.

The covalent binding of pyridoxal 5'-phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high-affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H] PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu-Asp-Glu-Leu-Arg-Asp-Glu-Gly-Xaa-Ala-Ser-Ser-Ala-Lys which corresponds to residues 182-195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys525) and acetylation by aspirin (Lys199).

Metabolism of pyridoxine and protein binding of the metabolites in human erythrocytes.

The uptake, distribution, and metabolism of pyridoxine in human erythrocytes were determined by incubating isolated erythrocytes in isotonic sodium phosphate, pH 7.4, with [3H]pyridoxine. After 60 min at 37 degrees C, the erythrocytes had taken up approximately 80% of the radioactivity. At least 99% of the radioactivity in the erythrocytes was in the supernatant fraction of the cells and nearly 80% of that radioactivity was in pyridoxal-phosphate and was protein bound. The B6-protein complex was stabilized by reduction with borohydride. To identify the protein to which the radioactive B6 was bound, the hemolysate supernatant was fractionated by chromatography on DEAE Sephadex and carboxy methyl-cellulose. The protein fractions containing radioactivity were analyzed further by chromatography on Sephadex G-150. Most of the radioactive B6 was found to Hb. Thus human erythrocytes rapidly took up pyridoxine and converted it to pyridoxal-phosphate. Much of the newly synthesized pyridoxal phosphate was bound to Hb.

Changes in blood lipid composition and response to interferon treatment in chronic hepatitis C.

To assess whether the initial status of lipid metabolism in patients with chronic viral hepatitis might correlate with outcome of therapy, 52 patients (32 males and 20 female) with chronic hepatitis C were studied: 44 were treated with human recombinant interferon-alpha 2b (3 MU three times per week for up to 12 months), and 8 served as controls. At baseline, sera were tested for total and HDL cholesterol, HDL2, HDL3, apolipoprotein A-I, apolipoprotein B, interferon-alpha, tumor necrosis factor, and interleukin-6. Changes in blood lipids were evaluated after 3, 30, and 90 days of treatment. HDL cholesterol, apolipoprotein A-I, and HDL3 decreased by 9.4-11.4% within 4 weeks of starting interferon treatment, but this effect was sustained only in patients with a primary response to interferon. On multivariate analysis, a primary response to interferon correlated with higher apolipoprotein A-I and lower (< 2.23 pg/ml) interleukin-6 levels (p < 0.005 for both). In contrast, a sustained response was significantly more common in patients with low (< or = 13.3 pg/ml) serum interferon-alpha and lower interleukin-6 at baseline but did not correlate with any of the blood lipids. Thus, in chronic hepatitis C, interferon treatment induces specific changes in blood lipids. The concentration of apolipoprotein A-I at baseline is a strong predictor of primary response to treatment, and the likelihood of sustained response seems to be reflected by lower cytokine activation.

Early carotid atherosclerosis in asymptomatic adults with primary moderate hypercholesterolemia: a case-control study.

It has been shown that severe hypercholesterolemia is associated with carotid atherosclerosis but it is unclear whether this is true for moderate hypercholesterolemia. The aim of the study was to determine the prevalence of ultrasound detectable extent and severity of carotid intima-media thicknesses in 143 asymptomatic (79 males, 64 females, age range 45-64 years) primary moderate hypercholesterolemic patients (serum LDL cholesterol range 160-190 mg/dl). This group was compared with 143 asymptomatic normolipidemic subjects (serum LDL cholesterol < or = 130 mg/dl and serum triglycerides < 200 mg/dl) matched for age, sex and other cardiovascular risk factors. The maximum intima-media thickness (IMT) was measured using B-mode ultrasonography at 12 sites on the near and the far wall of the common, bifurcation and internal carotid arteries. The mean-maximum IMT at the 12 sites was compared in cases and controls. Moreover, the prevalence of intima-media thickening (i.e. at least one of the 12 sites with an IMT equal to or greater than 1.0 mm but less than 1.3) and plaques (i.e. at least one of the 12 sites with an IMT equal to or greater than 1.3 mm) was considered in the two groups. The mean-maximum intima-media thickness was 0.97 +/- 0.12 mm in hypercholesterolemic patients and 0.93 +/- 0.05 mm in controls (P < 0.0001). Intima-media thickening and plaques were detected in 76% of hypercholesterolemics vs. 57% of controls (P < 0.0002). Gender did not influence these differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors associated with cognitive impairment among older Italian inpatients. Gruppo Italiano di Farmacovigilanza nell'Anziano (G.I.F.A.).

OBJECTIVE: To examine the association of cognitive impairment with educational, demographic, and nutritional variables in older hospitalized people. DESIGN: Survey of older patients admitted consecutively to a hospital during two 2-month periods in 1993. SETTING: Patients admitted for general medical care at 35 hospitals participating in the GIFA study throughout Italy. PARTICIPANTS: A total of 3628 patients aged 65 or older were studied. MEASUREMENTS: The Hodkinson Abbreviated Mental Test (HAMT) was used as a screening method to assess the patients' basic cognitive function. Multiple logistic regression was used to examine the association between cognitive impairment and demographic, educational or nutritional variables. RESULTS: Twenty-nine percent of older inpatients were classified as having cognitive impairment, with similar distribution of HAMT score found in both genders. Educational attainment has a highly significant inverse relationship with cognitive impairment (highest education: OR 0.32; 95% CI 0.20-0.52). Moreover, cognitive impairment decreased with increasing body mass index (3rd tertile: OR 0.69; 95% CI: 0.51-0.93), cholesterol serum level (highest values: OR 0.59; 95% CI 0.37-0.93), circulating lymphocytes (highest values: OR 0.55; 95% CI 0.45-0.69), and serum albumin (highest values: OR 0.60; 95% CI 0.47-0.76), with a gradient of influence for each variable. CONCLUSIONS: Educational attainment affects cognitive function in older inpatients. The strong association between cognitive impairment and nutritional variables suggests that every effort to improve nutritional status is needed in approaching cognitive impairment in older patients.

Insulin-like growth factor system gene expression in women with type 2 diabetes and breast cancer.

BACKGROUND/AIMS: A twofold increased risk for breast cancer has been reported recently for women with late onset diabetes. Most studies showed that there were differences in serum concentrations of insulin-like growth factors and related proteins between women with and without diabetes who have breast cancer. This study investigated the expression of these markers at the cellular level in a cohort of women with and without type 2 diabetes who underwent biopsy because of a breast lump. METHODS: Relative quantitative analysis of specific mRNA sequences was performed after extraction and reverse transcription polymerase chain reaction amplification from formalin fixed and paraffin wax embedded tissues. Sixty seven breast surgical specimens from women with and without diabetes who did not have cancer and from women with and without diabetes who did have cancer were studied for insulin-like growth factor I (IGF-I), the IGF-I receptor (IGF-IR), insulin-like growth factor binding protein 3 (IGFBP-3), and oestrogen receptor 1 gene expression. RESULTS: The expression of IGF-I and IGF-IR was significantly lower in the cancer groups, whereas there was no significant difference for IGFBP-3 between women with and without cancer. Moreover, there was a good correlation between the expression of IGF-I and IGF-IR in women without cancer: this link was still present in breast tissue from patients with diabetes and cancer, whereas it was lost in patients without diabetes but with cancer. CONCLUSIONS: These differences in IGF-I/IGF-IR expression could contribute to the increased risk for breast cancer in women with type 2 diabetes.

The effect of tyrosine-deficient total parenteral nutrition on the synthesis of dihydroxyphenylalanine in neural tissue and the activities of tyrosine and branched-chain aminotransferases.

The poor solubility of tyrosine (Tyr) limits the amount of this amino acid in total parenteral nutrition (TPN). In rats maintained on a standard pediatric TPN mixture, plasma and brain concentrations of Tyr are reduced to about 25% of the levels in chow-fed controls. To determine whether these low concentrations of Tyr affect the synthesis of catecholamines in neural tissue, the rate-limiting step (conversion of Tyr to dihydroxyphenylalanine [DOPA]) is studied by administering NSD-1015 to block the pyridoxal phosphate (PLP)-dependent decarboxylation of DOPA. However, in TPN rats, plasma concentrations of Tyr are increased by drug treatment. Because brain Tyr is also increased, these and other experiments using NSD-1015 clearly overestimate the rate of DOPA synthesis for drug-free rats on TPN. Nevertheless, in TPN rats, there is less DOPA in the brain in one experiment and less DOPA in the olfactory bulbs in another, versus control rats. Further examination of the metabolic effects of NSD-1015 reveals that the drug also elevates the concentration of branched-chain amino acids (BCAAs) in the plasma of TPN rats. These findings result from inhibition by NSD-1015 of the PLP-dependent aminotransferases that initiate catabolism of Tyr in the liver and BCAAs in the muscle. Despite the pronounced reduction in plasma Tyr, TPN rats showed a marked increase in the activity of hepatic Tyr aminotransferase compared with chow-fed controls. Conversely, although TPN elevates BCAA concentrations in plasma, the activity of branched-chain aminotransferase (BCAT) in the heart muscle of TPN rats is not different from control values. Different values but the same relationships are seen in drug-free rats.

Lipoprotein(a) serum concentration in familial combined hyperlipidemia.

The lipoprotein(a) (Lp(a) concentrations in serum were measured by an ELISA technique in 53 subjects affected by familial combined hyperlipidemia (FCHL) and in 347 healthy individuals. Lp(a) geometric means did not differ significantly between the two groups despite the different distributions. In hyperlipidemic subjects, the distribution was markedly shifted to the right (median 17 mg/dl) while in controls it was highly skewed to the left (median = 11 mg/dl). In FCHL, Lp(a) serum levels did not differ between patients with or without coronary heart disease (CHD). It was concluded that, differently from familial hypercholesterolemia (FH), in FCHL Lp(a) may not be elevated in comparison with an adequate control population.

Tyrosine fluorescence of two tryptophan-free proteins: histones H1 and H5.

The fluorescence intensity of the single tyrosine residue in histone H1 increases from RTYR = 0.3 to RTYR = 1.3 as the protein undergoes a conformational change from the random coil state to a folded form. Enhanced fluorescence in the folded state has not been observed before in ap protein. Histone H5 shows no change in fluorescence intensity on folding. This is interpreted as a result of compensation between enhanced and reduced fluorescence in the three tyrosine residues.

Intrinsic and extrinsic fluorescence of histones H2A and H2B: a conformational study.

Intrinsic and extrinsic fluorescence measurements suggest that H2A and H2B histones, in a partially secondary structure, self-aggregate into assemblies in which some tyrosine groups are buried in a hydrophobic environment and show enhanced fluorescence, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) indicates heterogeneity among the binding sites whose number depends on the pH values of the solutions. Warfarin, used as hydrophobic probe, shows that during the process of self-association and cross-complexing of the two histones there is the covering of some hydrophobic sites of the proteins.

Activation of coagulation by a LDL-apheresis device.

LDL-apheresis often induces an almost constant and progressive increase of the differential pressure of plasma flowing through the dextran sulphate cellulose column, reducing the efficacy of the treatment. On two occasions we were able to identify a fibrin plug by immunofluorescence. Our aim was to verify the modification of some coagulation indicators in patients undergoing LDL-apheresis and whether an activation of coagulation occurs in the LDL-apheresis device. Blood samples were obtained from six patients with familial hypercholesterolaemia who were undergoing LDL-apheresis. During the same session further blood/ plasma samples were taken from the LDL-apheresis device at different sites and at different volumes of filtered blood. In patients after LDL-apheresis the following modifications were found: a 25% decrease of fibrinogen and a slight increase in F1 + 2 plasma levels. No relevant changes in thrombin-antithrombin complexes and fibrinopeptide A plasma levels were noted. In the LDL-apheresis device the main results were: (a) fibrinogen was trapped in the dextran sulphate cellulose column in the early phases; (b) activation of coagulation was recognisable in the plasma separator during the procedure and progressively increased with duration of LDL-apheresis; (c) thrombin-antithrombin complexes, formed in the plasma separator, were retained by the dextran sulphate cellulose column. In conclusion, LDL-apheresis activates coagulation in the device. Shortening cycle time or using nafamostat mesilate as an anticoagulant, could be interesting alternatives for improving the procedure.

Low density lipoprotein-apheresis decreases oxidized low density lipoproteins and monocyte adhesion to endothelial cells.

The mutual interaction between monocytes and low density lipoprotein (LDL) in atherogenesis prompted a test of the hypothesis that LDL-apheresis could reduce the adhesive properties of monocytes to endothelium; and therefore interfere with a key mechanism in atheroma formation. Five patients affected by heterozygous familial hypercholesterolemia were studied. All patients received LDL-apheresis treatment with selective adsorption of LDL-cholesterol on dextran-sulphate columns. Low density lipoprotein particles were isolated by sequential preparative ultracentrifugation and subfractionated by ion exchange high performance liquid chromatography. Thiobarbituric acid reacting products of lipid peroxidation were measured fluorometrically. Vitamin E was estimated by high performance liquid chromatographic technique. Monocytes were isolated from patients blood before and 1 day after LDL-apheresis by Percoll gradient. The blood samples for monocyte adhesion were drawn from control subjects for 2 consecutive days. The adhesion of monocytes to an endothelial monolayer was evaluated by assaying the peroxidase content of the adherent monocytes. Low density lipoprotein-apheresis reduced total cholesterol (-65%; p < 0.01), LDL-cholesterol (-75%; p < 0.01), triglycerides (-51%; p < 0.05), and fibrinogen (-28%; p < 0.01). With LDL-apheresis treatment, a reduction of 54% in oxidized LDLs was observed; vitamin E concentration significantly increased in LDLs (+ 14.2%; p < 0.05). The monocyte adhesion decreased by approximately 61% after apheresis; the variation became statistically significant (-65%; p < 0.01) when endothelial cells were stimulated by lipopolysaccaride.

Carotid atherosclerosis: echographic patterns versus histological findings.

The study was carried out on 25 whole carotid arteries explanted from a corpse and perfused at constant pressure to reproduce the conditions of an in vivo examination as much as possible. Out of 5 samples with intimal thickening detected by echo, fibrosis of the tunica media was observed by the pathologist in 4 and microcalcification in 1. In 4 vessels with soft plaques at echo scanning, a wide necrotic area (2 cases), slack connective tissue (1 case) and cystic lesions (1 case) were observed. Hard lesions with (5 cases) or without (2 cases) a cone of shadow at echo evaluation corresponded to fibrous (2 cases) or fibrocalcific (3 cases) plaques. The histological study of the two echo-diagnosed thrombi showed an intermediate echographic pattern and the main feature of the non-occluding thrombus was the absence of a lumen-lesion interface. Mixed plaques were diagnosed at echo in 9 arteries and the correspondent histological aspect was a typical atheromatous lesion in all cases. Thus, the comparison of the ultrasound image with the histological findings proved the reliability of echography in the detection of atheromatous lesions with an excellent agreement between the results at the 2 examinations. Since the type of carotid lesions has an impact upon clinical events these results might support the use of vascular ultrasound images in clinical applications.

[Hypothyroid myopathy in a young adult]. / Miopatia ipotiroidea in un giovane adulto.

We describe here the case of a young man admitted to our department for asthenia and diffuse myalgia. Because of persistently increasing aminotransferase levels over the preceding 2 years, he had undergone a number of diagnostic procedures to evaluate liver function. On admission to our department, the patient suffered from asthenia, myalgia, notable hair loss and sinus bradycardia. Laboratory examinations confirmed severe primary hypothyroidism with high serum levels of aminotransferase, aldolase and creatine-phosphokinase attributable to thyroprivic myopathy. The thyroid deficit was caused by autoimmune atrophic thyroiditis. Treated with levothyroxine, the patient recovered fully from his illness, his normal metabolic state was restored, and his serum aminotransferase level returned to normal.