Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry.

D-lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased D-lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects D-lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect D-lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of L- and D-lactic acid by a two-step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic™ R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2-400 and 0.5-100 µmol/L respectively for L- and D-lactic acid. The limit of detection of D-lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis.

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase.

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.

Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I.

It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) beta1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC beta1. In cells overexpressing a PLC beta1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I.

Preexercise glucose ingestion and glucose kinetics during exercise.

The present study was undertaken to examine the effects of glucose ingestion before exercise on liver glucose output and muscle glucose uptake during exercise. On two occasions, at least 1 wk apart, six trained men (peak pulmonary O2 uptake = 5.11 +/- 0.17 l/min) ingested 400 ml of a solution containing either 75 g glucose [carbohydrate (CHO)] or a sweet placebo [control (Con)] 30 min before 60 min of exercise at 71 +/- 1% peak pulmonary O2 uptake. Glucose kinetics (rates of appearance and disappearance) were measured by a primed continuous infusion of [6,6-2H2]glucose. Liver glucose output was derived from total glucose appearance and the appearance of ingested glucose from the gut. After glucose ingestion, plasma glucose increased to 6.4 +/- 0.4 mmol/l immediately before exercise, fell to 4.2 +/- 0.5 mmol/l after 20 min of exercise, and then increased to a higher value than in the Con group (5.4 +/- 0.3 vs. 4.7 +/- 0.1 mmol/l; P < 0.05) after 60 min of exercise. In the CHO group, plasma insulin was higher immediately before exercise (P < 0.05) and, despite falling during exercise, remained higher than in the Con group after 60 min of exercise (57.0 +/- 11.4 vs. 24.8 +/- 1.7 pmol/l; P < 0.05). The rapid fall in plasma glucose in the CHO group was the result of a higher muscle glucose uptake with the onset of exercise (P < 0.05), which could not be matched by the glucose rate of appearance. Liver glucose output was decreased by glucose ingestion, and although it increased during the early stages of exercise in the CHO group, it did not rise above the basal values and was reduced by 62% over the 60 min of exercise compared with the Con group. In summary, preexercise glucose ingestion results in increased muscle glucose uptake and reduced liver glucose output during exercise.