Ajit Sanghvi

Effects of casein and soy protein on hepatic and serum lipids and lipoprotein lipid distributions in the rat

Rats fed a semipurified diet containing casein developed higher levels of circulating triglycerides and cholesterol than animals fed a soy protein-containing diet. The increased serum lipid levels in non-fasted rats were associated largely with the d less than 1.006 g/ml lipoprotein particles (e.g. chylomicrons or very low density-like lipoproteins). In addition, casein-fed rats exhibited higher levels of circulating insulin and depressed hepatic 7 alpha-hydroxylase levels compared to soy-fed rats. Supplementation of the casein diet with arginine, to give an arginine/lysine ratio comparable to that in the soy diet, resulted in a reduction of d less than 1.006 g/ml lipids, a reduction in serum insulin levels and an elevation in hepatic 7 alpha-hydroxylase activity. Supplementation of the soy diet with lysine also resulted in modification of these parameters toward those observed with casein diets, albeit the effects were less dramatic. The results suggest that the hyperlipidemia associated with feeding casein-based diet is associated with decreased rates of clearance of chylomicron-like lipoproteins and their component triglycerides and cholesterol. Furthermore, this is largely prevented by addition of arginine to diets containing casein as the sole protein source.

Reversible activation-inactivation of cholesterol 7α-hydroxylase possibly due to phosphorylation-dephosphorylation

Abstract The activity of microsomal cholesterol 7α-hydroxylase is shown to be increased in vitro by ATP, Mg 2+ , and a cytosolic protein fraction. There was a loss of enzyme activity in the presence of E. coli alkaline phosphatase which was proportional to the amount of phosphatase. Much of this loss was recovered upon addition of ATP, Mg 2+ , and a cytosolic protein fraction.

Cyclosporine Kinetics in Healthy Volunteers

The pharmacokinetics of cyclosporine was studied in five healthy male volunteers following intravenous administration. The subjects received 2.1 mg/kg of cyclosporine as a two‐hour intravenous infusion. Blood samples were collected over the subsequent 48 hours. Cyclosporine was extracted from whole blood and analyzed by high‐performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Following the intravenous infusion of cyclosporine, the drug exhibited multicompartmental behavior. The harmonic mean distribution half‐life based on HPLC data was 0.45 hours, and the harmonic mean terminal disposition half‐life was 6.2 hours. The clearance of cyclosporine based on HPLC cyclosporine concentrations was 3.9 mL/min/kg, and the volume of distribution at steady state of cyclosporine was 1.23 L/kg. Cyclosporine has a shorter half‐life, lower clearance, and smaller Vss in healthy persons as compared to patient populations. The differences observed in the pharmacokinetics of cyclosporine in healthy persons as compared to patient populations may be due to differences in hematocrit, lipoprotein profiles, and/or concurrent drug therapy between the groups. Cyclosporine concentrations determined by RIA were consistently higher than those determined by HPLC, resulting in a significantly higher area under the blood concentration versus time curve and lower clearance rate for cyclosporine. We conclude that: (1) kinetic parameter estimates for cyclosporine are different in healthy individuals as compared with organ‐transplant recipients, and (2) the kinetic parameters for cyclosporine are different, depending on the assay technique used.

CYCLOSPORINE METABOLISM AND PHARMACOKINETICS FOLLOWING INTRAVENOUS AND ORAL ADMINISTRATION IN THE DOG

The influence of assay method on single dose cyclosporine (CsA) pharmacokinetics was studied in nine dogs receiving either i.v. or oral CsA. Samples were drawn from hepatic, portal, and systemic veins at various times after the dose and CsA levels were determined by radiommunoassay (RIA) and high-performance liquid chromatography (HPLC). Blood concentration-time data were analyzed by nonlinear least-squares regression, using two-compartment models. RIA/HPLC ratios for all samples were greater than one, and did not change significantly over time. The mean RIA/HPLC ratios for samples drawn from all three veins were higher after oral than i.v. doses of the drug (P<0.05). Area under the concentration-time curve (AUC) was higher and systemic clearance (Cls) lower than calculated on the basis of RIA results, regardless of the route of administration. AUC calculated for CsA metabolites (RIA-HPLC) was highest in the portal vein after an oral dose of CsA. Bioavailability was 20.4% and 27.0% when estimated using HPLC and RIA data, respectively. The mean CsA metabolite index (CMI), when calculated for hepatic, portal, or systemic vein, was greater when the drug was administered orally. The mean hepatic extraction ratio (HER) of the parent drug and for CsA metabolites was approximately 23% in i.v. and p.o. studies. These results suggest that the gastrointestinal tract may play a role in the metabolism of CsA when the drug is administered orally. In addition, if CsA metabolites not measured by HPLC have either toxic or immunosuppressive properties, the RIA assay may be more useful for monitoring patients.

Fk506: A novel immunosuppressive agent characteristics of binding and uptake by human lymphocytes

Several studies indicate FK506, a novel compound (Fig. 1), to be a potent immunosuppressive agent (1). Like cyclosporine, FK506 is isolated from a fungus, Streptomyces tsukubaensis, and is hydrophobic. Although cyclosporine is a peptide of larger molecular weight than FK506, both are cyclic compounds, with cyclosporine being more rigid than FK506. Recent in vitro studies suggest that FK506 possesses immunosuppressive properties similar to those of cyclosporine, implying that the mechanism of action for these two drugs may be similar as well. The immunosuppressive effect of FK506 is several-hundred–fold greater than that of cyclosporine (2, 3). As previously demonstrated for cyclosporine, the inhibitory effect of FK506 is seen in mixed leukocyte culture and on secondary proliferation of alloreactive T cells harvested from MLC or propagated from organ transplant biopsies (4). The published data further show that immunosuppression by FK506 may be mediated through an inhibition of interleukin-2 release (2, 4), again emphasizing the similarities between cyclosporine and FK506. FIGURE 1 FK506. In this report we describe the characteristics and kinetics of cellular uptake and intracellular binding of FK506 by human peripheral blood lymphocytes. PBL were isolated from the blood of four healthy donors and depleted of their monocyte content as described previously (5). Binding and uptake of FK506 by PBL is shown in Figure 2. The uptake of FK506 by PBL is a saturable process, with saturation occurring at an approximately 0.5 µM concentration of the drug. Scatchard analysis of the binding data is consistent with two distinct classes of binding sites—one with a Kd = 3.9±1.8×10−8 M for the high affinity sites, and the second with a Kd = 5.2±0.8×10−6 M for the low affinity sites. This analysis further reveals that there are 5.6±1.0×l04 and 2.5±0.9×106 high- and low-affinity binding sites, respectively, per cell. LeGrue et al. (7) have reported that binding of cyclosporine by normal PBL also exhibits two classes of binding sites with a Kd of 2–6×10−9 M for the high-affinity site and a Kd of about 10−7 M representing a low-affinity site. They have further indicated that only B cells possess high-affinity sites. Our data, together with this information, seem to further delineate the similarities between FK506 and cyclosporine. FIGURE 2 Binding of FK506 by human peripheral blood lymphocytes. Normal human lymphocytes (5×106) were incubated for 60 min at 37°C in 250 µl of 1% BSA-RPMI 1640 medium containing the indicated concentrations of FK506. The cells were chilled ... Merker and Handschumacher (8) have studied the intracellular localization of cyclosporine by a murine thymoma cell line (BW5147), and have shown that cyclosporine binds to a cytosolic protein (termed cyclophilin) with an apparent molecular weight of 15,000–20,000 daltons. More recently, Fabre et al. (9) have shown the presence of a similar protein in a human Burkitt lymphoma cell line (RAJI cells). The dissociation constant for the binding of cyclosporine to cyclophilin has been reported to be approximately 2.2 µM. We have investigated the binding of FK506 to cytosolic proteins obtained from PBL. The elution profile of FK506 and cyclosporine on a Bio-Gel P-60 exclusion column that had been calibrated with ovalbumin, carbonic anhydrase, cytochrome C, and vitamin B12 is shown in Figure 3. A graph of the log molecular weight versus the ratio of the elution volume to the void volume for the molecular weight markers is shown in the insert. These data suggest that both FK506 and cyclosporine elute in association with a protein with an apparent molecular weight of approximately 18,000–19,000 daltons. Whether this is incidental or there is in fact only one protein that binds both drugs is not clear at present. But, if a single protein, such as cyclophilin, is involved in the binding of both these drugs, then the question of whether two separate binding sites are involved in this process remains to be investigated. Recently, we have demonstrated that human PBL exposed to FK506 for periods varying from 1 hr to 40 hr are able to take up about 20% more cyclosporine relative to control cells not exposed to FK506 (10). Similarly, FK506 exposed cells show considerably increased sensitivity to cyclosporine (2–4) in terms of mixed lymphocyte reaction and primed lymphocyte tests. This evidence seems to suggest that binding of one drug, for instance FK506, modifies the cellular response to another drug (cyclosporine), resulting in a greater uptake of cyclosporine by the cell. FIGURE 3 Elution profile of the intracellular proteins of PBL on Bio-Gel P-60. Normal human lymphocytes (100×106) cells were incubated for 60 min at 37°C in 4 ml 1% BSA-RPMI 1640 medium containing 2 µg FK506 and 1.5 µg (3H) cyclosporine ... In summary, kinetics of binding and uptake of FK506 by PBL, and the apparent common association of FK506 and cyclosporine with an intracellular protein, enhances further the similarities between these two immunosuppressive agents.

Isolation and identification of a mevalonolactone by-product formed during the assay for 3-hydroxy-3-methylglutaryl coenzyme a reductase activity

Abstract A mevalonolactone by-product formed during the assay for 3-hydroxy-3-methylglutaryl-CoA reductase activity is isolated and proof of its structure is provided from gas chromatographic, NMR and mass spectrometric studies.

Effect of detergents on in vitro 7α-hydroxycholesterol formation by rat liver microsomes

Formation of 7α-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7α-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7α-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7α-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (Ks>Kp). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.

On the differential suppression of cholesterol synthesis by low density lipoprotein in B and T lymphocytes

Cholesterol synthesis and its suppression by low density lipoprotein cholesterol were measured in purified B and T peripheral blood lymphocytes. After preincubation for 53 h in lipoprotein-deficient serum, both B and T cells exhibited increased cholesterol synthesis as compared with synthesis measured in cells immediately after their isolation from blood and without preincubation with lipoprotein-deficient serum. The magnitude of this increase was far greater in T cells in comparison with that in B cells in all subjects studied. But, whereas there was an immediate and progressive suppression of cholesterol synthesis in lipoprotein-deficient serum-incubated T cells as the concentration of low density lipoprotein cholesterol in the medium was increased, synthesis in lipoprotein-deficient serum-incubated B cells remained insensitive to the presence of low density lipoprotein in the medium. Hydroxymethylglutaryl-CoA reductase activity was observed also to follow a similar pattern in both cell types. These observations may imply that one or more events, including binding of low density lipoprotein to its receptor, internalization and degradation of low density lipoprotein receptor complex finally leading to suppression of hydroxymethylglutaryl-CoA reductase activity and cholesterol synthesis, fail to take place in B cells.

Regulation of bile acid synthesis: isolation and characterization of microsomal phosphatases

In preliminary experiments, we have shown that rat liver microsomes possess phosphatase activity which was inhibited in the presence of sodium fluoride. We have now separated six microsomal phosphatase fractions appearing to be isoenzymes. They all possess different kinetic constants and are not equally inhibited by tartrate and fluoride ions, inhibitors of phosphatase activity. One phosphatase fraction, in fact, is almost completely unaffected by fluoride ion. More pertinent to our interest, these isoenzymes exhibit differing abilities to modulate the activities of hydroxymethylglutaryl CoA reductase, acyl-CoA:cholesterol O-acetyltransferase, and cholesterol 7 alpha-hydroxylase. Interaction of four of the fractions with rat liver microsomes resulted in a decrease in cholesterol 7 alpha-hydroxylase activity; two were without effect.

Estriol determination in pregnancy urine by gas chromatography

Abstract Determination of free estriol in pregnancy urine by gas chromatography is described and data are presented to support the accuracy of this procedure as compared to the gas chromatography of estriol triacetate. Data from infrared and nuclear magnetic resonance spectroscopy and from mass spectrometric studies on estriol and estriol triacetate obtained after passage through gas Chromatographic columns indicate no structure modifications in these compounds.