Simultaneous measurement of phenylalanine and tyrosine in phenylketonuric plasma and dried blood by high-performance liquid chromatography.

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with alpha-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm x 4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.

Insulin-like growth factor-I treatment to enhance renal function in advanced chronic renal failure.

Because of the potential for IGF-I to enhance renal function in advanced chronic renal failure (CRF) we set out to determine whether IGF-I can induce a sustained increase in renal function in patients with near end-stage renal failure. To this end we first examined the impact of CRF on the pharmacokinetics of IGF-I and then we examined the effect of prolonged IGF-I treatment on the renal function of patients with an average GFR of 17 mL/min/1.73 m2. Interestingly the metabolic clearance rate of IGF-I in CRF subjects was similar to that in normal subjects even though the total serum IGF-I levels rose to higher maximum levels. This increase was due to a reduced volume of IGF-I distribution, a consequence of the elevated serum IGF binding proteins in CRF subjects. Treatment with IGF-I (60 mg/kg twice daily sc) for 31 days resulted in a 14% and 18% increase in the inulin and PAH clearances respectively (n = 6 patients). These parameters returned to basal levels on stopping treatment. Serum immunoreactive IGFBP-3 levels fell and IGFBP-2 and -3 levels rose during IGF-I therapy. Adverse effects were mild, of short duration and easily manageable. Thus IGF-I pharmacokinetics are largely unchanged in CRF and the administration of IGF-I produces a modest improvement in the GFR. These results appear to justify more extensive examination of the therapeutic role of IGF-I in the treatment of CRF.

Early experience with extended use of insulin-like growth factor-1 in advanced chronic renal failure.

Short-term high-dose insulin-like growth factor-1 (IGF-1) therapy has been shown to enhance glomerular filtration rate (GFR) in end-stage chronic renal failure (CRF), but the efficacy and safety of prolonged therapy is unproven. To determine if prolonged therapy with IGF-1 can enhance renal function in advanced CRF, eight patients were entered into a study to receive one month of IGF-1 treatment, 60 micrograms/kg subcutaneously b.i.d. Six patients completed the study and two dropped out for reasons considered to be unrelated to the IGF-I treatment. Baseline inulin and PAH clearances averaged 17 +/- 3 and 66 +/- 14 ml/min/1.73 m2, respectively, in the subjects who completed the study. With treatment there was a modest 14% increase in the average GFR which approached statistical significance (P = 0.051). After stopping treatment the clearance values returned to basal values. The PAH clearance showed a similar trend. There were no significant changes in BUN, serum creatinine or electrolyte levels. On the other hand there were marked changes in the serum IGF binding protein (IGFBP) profile. Serum IGFBP-3 levels fell while IGFBP-1 and -2 levels rose during treatment, changes that likely affect the bioavailability of IGF-I. Thus, in this small series of patients IGF-1 treatment produced significant changes in the serum IGFBP profile and a modest upward trend in the GFR.

Pharmacokinetics of insulin-like growth factor-1 in advanced chronic renal failure.

Information regarding the impact of chronic renal failure (CRF) on IGF-1 serum clearance is limited. Thus we evaluated the pharmacokinetics of insulin-like growth factor-1 (IGF-1) in six normal adults and six adults with advanced CRF (serum creatinine 7 +/- 0.8 mg/dl). All subjects were given 80 micrograms/kg recombinant human IGF-1 s.c. and blood was sampled over 48 hours. Baseline total serum IGF-1 levels were similar in both groups, but peak levels were elevated significantly in CRF; this was apparently related to the reduced distribution volume in CRF subjects. CRF did not affect the metabolic clearance rate (MCR) of total serum IGF-1. Immunoreactive IGF binding protein-3 (IGFBP-3) levels were greater in CRF. Western immunoblots revealed that the apparent increase in IGFBP-3 was largely due to an increase in immunoreactive fragments. IGFBP-3 protease activity was not increased. Thus IGFBP fragment accumulation likely reflects reduced fragment clearance. Western ligand blots revealed elevated 30 and 34 kDa IGFBP levels and IGFBP products in CRF serum. Serum acid labile subunit levels were unchanged in CRF. Peak free IGF-1 levels and the MCR of free IGF-1 did not differ between groups. In both groups the MCR of free IGF-1 exceeded the MCR of total IGF-1 by approximately 30-fold. These data suggest that in CRF patients receiving s.c. IGF-1: (a) total serum IGF-1 levels are increased as a result of elevated circulating IGFBPs that may restrict the distribution of IGF-1 beyond plasma; (b) serum free IGF-1 levels are not altered; and (c) the IGF-1 MCR is unchanged in CRF. Thus, in advanced CRF, apart from a reduction in the total IGF-1 volume of distribution the pharmacokinetics of IGF-1 are largely unaltered.

Effects of Mode of Sterilization on the Recovery of Phenolic Antioxidants in Laboratory Media Assessed by Nonderivatizing Gas Chromatography-Mass Spectrometry ‡.

The effects of autoclaving and filter sterilization on the recovery of butylated hydroxyanisole, butylated hydroxytoluene, tertiary butylhydroquinone, and propyl gallate in laboratory media were investigated by a nonderivatizing gas chromatography-mass spectrometry (GC-MS) method. Lauryl tryptose broth (LTB) or brain heart infusion (BHI) broth were treated with the combinations of the phenolic antioxidants at 200 ppm. The antioxidants were dissolved in 95% ethanol and either added directly to the media followed by autoclaving or filter-sterilized and then added to sterilized media in a flask. Results suggested that antioxidant recoveries were affected by the modes of sterilization as well as by the complexity of the medium. More antioxidants were recovered from the filter-sterilized than from the autoclave-sterilized samples for LTB medium, but no clear difference was seen for BHI broth. The recovery was lowest with butylated hydroxytoluene regardless of medium type or sterilization mode used. The extraction procedure applied as well as the nonderivatizing GC-MS method appeared to be adequate for the simultaneous determination of the four antioxidants.

Developmental switch in the hippocampal serotonin receptor linked to phosphoinositide hydrolysis.

5-HT2A and 5-HT2C receptors couple to the phosphoinositide hydrolysis signal transduction pathway. The present pharmacological analyses provide evidence for a switch in the functional 5-HT receptor in rat hippocampus (from 5-HT2A to 5-HT2C) between the first and third weeks of life. Spiperone and MDL 100,507, antagonists that bind with 300- to 1000-fold higher affinity to 5-HT2A receptors, blocked 5-HT-induced phosphoinositide hydrolysis in hippocampi of 7-day-old, but not 21-day-old, rats. In contrast, the non-selective 5-HT2A/2C receptor antagonists, mesulergine and mianserin, blocked 5-HT-mediated phosphoinositide hydrolysis in both 7- and 21-day-old rats. These results suggest that the 5-HT-induced phosphoinositide hydrolysis signal in hippocampus of 7-day-old rats is mediated predominantly by 5-HT2A receptors, while in 21-day-old rats the phosphoinositide hydrolysis signal is mediated in large part by 5-HT2C receptors. Neither 5-HT2A or 5-HT2C receptor mRNA nor the binding site densities of the two receptors were altered between the two ages, ruling out developmental changes in receptor density as an explanation for the observed differences. We conclude therefore that the hippocampal 5-HT receptor that links to phosphoinositide hydrolysis switches during postnatal development of rats, perhaps reflecting differences in the coupling of 5-HT2A and 5-HT2C receptors to intracellular effector molecules.

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.