Intrapersonal HbA(1c) variability and the risk of progression of nephropathy in patients with Type 2 diabetes.

AIM: To investigate the association between nephropathy and HbA(1c) variability (assessed as the standard deviation of each patient's HbA(1c) measurements) among patients with Type 2 diabetes. METHODS: Albumin excretion rate and HbA(1c) were measured in 2103 patients followed up for a mean 6.6 years. Multivariate Cox regression analysis was used to determine the influence of HbA(1c) variability on the risk of progression of nephropathy after adjustment for age, sex, duration of diabetes, baseline condition (two cohorts defined by duration of diabetes, retinopathy and albumin excretion rate), baseline HbA(1c) , insulin use, BMI, use of anti-hypertensive agents, smoking, lipid status, retinopathy, updated mean HbA(1c) and number of HbA(1c) measurements. RESULTS: Nephropathy progressed in 18.3% of subjects. HbA(1c) variability was significantly greater among progressors than among non-progressors (12 vs. 10 mmol/mol; 1.12 vs. 0.90%; P < 0.0001) and was a significant predictor of progression of nephropathy even after adjustment for updated mean HbA(1c) and other risk factors (hazard ratio 1.37, 95% CI 1.12-1.69). CONCLUSION: In patients with Type 2 diabetes, the risk of progression of nephropathy increases significantly with HbA(1c) variability, independently of the influence of updated mean HbA(1c) .

Therapeutic criteria in communicating childhood hydrocephalus.

AIM: The aim of this study was to evaluate the usefulness of cerebral blood flow velocity in the middle cerebral artery measured by transcranial Doppler as criteria to therapeutic action in communicating hydrocephalic children. METHODS: In eight non-tumoral communicating hydrocephalic infants, ranging from five to 18 months of age, monitored from 18 to 36 months (mean time of follow-up: 24.25 months), cerebrospinal fluid (CSF) oxypurines (hypoxanthine and xanthine) and uric acid levels were compared by means of the Evans' index, the mean weekly increase in cranial circumference, and the transcranial Doppler measurements. RESULTS: Results indicate that clinical (mean weekly increase in head circumference), radiological (Evans' index), biochemical (oxypurines and uric acid in the CSF), and hemodynamic (transcranial Doppler) criteria have the same role in monitoring infantile hydrocephalus. CONCLUSION: In conclusion the transcranial Doppler measurement can be done noninvasively and examinations can be repeated when needed, obtaining immediate RESULTS: Hence, it is the most adequate monitoring technique in clinical practice.

Corrected Fructosamine improves both correlation with HbA1C and diagnostic performance.

AIMS: There is increasing interest in using fructosamine measurements in screening for or managing diabetes, yet uncertainty remains as to whether these measurements should be corrected for variation in serum protein concentrations. METHODS: We considered all sets of simultaneous measurements of fructosamine, albumin, total serum protein (TP), fasting plasma glucose (FPG) and HbA1C recorded in our laboratory over 10years. The relationships between fructosamine and other variables were studied by multivariate linear regression and other analyses, and receiver operating curves (ROCs) were analysed to compare the diabetes screening performance of uncorrected fructosamine to those of albumin-corrected fructosamine (FAAlb) and TP-corrected fructosamine (FATP). RESULTS: 40,938 sets of measurements were collected from 20,114 patients. Though correlation between fructosamine and serum proteins was strongest among patients with HbA1C <6.5% (48mmol/mol), it was also significant in the whole sample (r=0.193 for albumin, r=0.213 for TP). With diabetes defined by HbA1C ≥6.5% (48mmol/mol), the areas under the ROCs of FAAlb (0.905) and FATP (0.895) were both significantly greater (P<0.001) than that of uncorrected fructosamine (0.878). Correction of fructosamine for albumin or TP slightly improved its correlation with HbA1C. There was no correlation of protein (albumin or TP) with log(fructosamine/protein). CONCLUSIONS: Fructosamine concentration correlates significantly with albumin and total protein concentrations throughout their ranges. Correction of fructosamine improves its correlation with HbA1C and its performance in detecting diabetes.

Application of enzyme preparations for extraction of berry skin phenolics in withered winegrapes.

Postharvest withering of grapes strongly affects the content and extractability of phenolic compounds in the production of sfursat, fortified and passito wines. This work evaluated the effectiveness of enzymes applied individually and/or in multi-enzyme blends, on the extraction of anthocyanins, oligomeric flavanols and polymeric flavanols from withered grape skins during simulated maceration. The study was performed on Vitis vinifera L. cv. Nebbiolo and Barbera because of their different skin phenolic profile and cell wall composition. Our findings highlight that the relationship between skin mechanical properties (berry skin break force and energy) and extraction yield of phenolic compounds is variety dependent. Significant correlations were found between the skin softening associated with cell wall degradation and the extraction of anthocyanins and flavanols in Nebbiolo, for which polygalacturonase, individually or in multi-enzyme blends, plays an important role. In Barbera, the extractability of phenolic compounds was not affected by the presence of exogenous enzymes.

Alteration of renal carnitine metabolism by anticonvulsant treatment.

We administered therapeutic doses of valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHT), and phenobarbital (PHB) to mice for 7 days, and 8 hours after the final dose we measured the concentrations of carnitine in serum, liver, kidney, skeletal muscle, and heart, and in the 7 days' accumulated urine. The results for serum and urine show that VPA induced a significant increase in renal clearance of acylcarnitine without affecting that of free carnitine, whereas CBZ, PHT, and PHB significant increased clearance of free carnitine but not that of acylcarnitine. Thus, VPA appears to reduce tubular resorption of acylcarnitine, and CBZ, PHT, and PHB appear to reduce tubular resorption of free carnitine.

Short-term effects of administration of anticonvulsant drugs on free carnitine and acylcarnitine in mouse serum and tissues.

1. The short-term evolution of concentrations of free carnitine and acylcarnitine was studied in the serum, liver, kidney, heart and skeletal muscle of mice after administration of single therapeutic doses of the anticonvulsant drugs, valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHT) and phenobarbitone (PHB). 2. The effects of the drugs were immediate but transitory, control levels of free carnitine and acylcarnitine having been recovered or almost recovered in serum and in all tissues 8 h post administration (p.a.). 3. VPA was the only drug that significantly reduced free carnitine concentration in serum, which recovered control levels by 4 h p.a. 4. All the drugs studied brought about marked deficits of serum acylcarnitine, which had disappeared 2 h p.a. in the case of VPA and not until 8 h p.a. for CBZ, PHT or PHB. 5. The minimum concentrations of free carnitine and acylcarnitine in serum were invariably associated with the maximum concentration of drug in serum. 6. Free carnitine concentration was not affected by VPA in any tissue, PHT and PHB brought about significant deficits in heart and kidney, and CBZ a significant deficit in muscle. 7. Acylcarnitine concentration was significantly reduced in heart, kidney and muscle by CBZ, PHT and PHB, but in liver the effects of all drugs were very small. 8. These results are compatible with the hypothesis that the primary cause of anticonvulsant-induced alteration of carnitine metabolism is interference with renal reabsorption of carnitine.

Effects of acute valproate administration on carnitine metabolism in mouse serum and tissues.

Carnitine concentrations in serum, liver, kidney, muscle and heart were determined 30 min, 2 hr and 4 hr after administration of single 50 mg/kg doses of valproic acid (VPA) or octanoic acid (OTA) of fasting mice. Half an hour post-administration (p.a.) of VPA, free carnitine concentrations were smaller than in controls in serum, liver, kidney and heart. Four hr p.a., the effects of VPA had disappeared from all the carnitine sources, which now had concentrations that were not significantly different from those of controls. The effects of OTA are different from, and sometimes the opposite of, those of VPA, showing that the effects of VPA are specific to it. Hyperammonemia, on the other hand, was greatest 4 hr p.a. of VPA. These findings show that the effect of VPA on carnitine metabolism is immediate but transient, and accordingly suggest that the carnitine deficiency observed in patients under prolonged treatment with VPA-containing anticonvulsants must be due to a more complex mechanism than direct interaction between carnitine and VPA.

Ghrelin-induced growth hormone secretion in humans.

Ghrelin is a novel growth hormone (GH) releaser acylated peptide that has recently been purified from stomach, and which potently binds to the GH secretagogue receptor. Ghrelin releases GH in vitro and in vivo in animal models, however its actions, potency and specificity in humans are unknown. In the present study, 12 healthy subjects were studied: 6 underwent four tests with ghrelin administered i.v. at the dose of 0 (placebo), 0.25, 0.5 and 1 microg/kg which corresponds to 0, 18, 37 and 75 microg total dose. A further 6 volunteers underwent two tests on different days with ghrelin at the dose of 3.3 or 6.6 microg/kg which corresponds to 250 microg and 500 microg total dose. Ghrelin-mediated GH secretion showed a dose-response curve, in which 1 microg/kg was the minimally effective dose in some individuals, but not as a group. On the contrary, the total doses of 250 microg and 500 microg elicited a powerful GH secretion, with a mean peak of 69.8+/-9.2 microg/l and 90.9+/-16.9 microg/l respectively, and areas under the curve of 4435+/-608 and 6125+/-1008 microg/l per 120 min respectively. All of them statistically significant vs placebo and vs the 1 microg/kg dose. Ghrelin administration also elicited a relevant dose-response mediated prolactin secretion suggesting no specificity of its actions. No relevant side effects were observed with ghrelin apart from a hyperhydrosis episode in two individuals tested with the higher ghrelin doses. In conclusion, ghrelin is a potent releaser of GH in normal individuals, with a dose-response pattern of operation. No saturating dose was observed.

Cord serum gamma glutamyltransferase in newborns.

This article reports correlations among gamma-glutamyltransferase (GGT), fetal haemoglobin (fH), alpha-fetoprotein, 5'-nucleotidase, ceruloplasmin, and direct, indirect, and total bilirubin in the serum of blood taken from the umbilical cords of 128 newborns delivered after 37-42 weeks of gestation. GGT was significantly correlated with alpha-fetoprotein, but not with direct bilirubin, indirect bilirubin, total bilirubin, fH, or %fH. Neither fH nor %fH were correlated with alpha-fetoprotein, but there was highly significant negative correlation between both fH and %fH on the one hand, and gestational age and weight at birth on the other. The %fH was also correlated negatively with ceruloplasmin, which in turn exhibited negative correlation with alpha-fetoprotein. The predominant forms of GGT in umbilical cord and adult sera were, respectively, those with alpha 1 and alpha 2 mobility. In cord sera, delipidation with n-butanol brought about loss of GGT activity and a shift from an alpha 1 to an alpha 2 position, whereas no significant effect of this kind was observed in adult sera. Affinity chromatography through Concanavalin A-Sepharose showed cord sera to contain a proportion of bound-GGT (68.5 +/- 5.5%) that was significantly greater (p less than 0.001) than that found in adult sera (59.8 +/- 10.2%). It is concluded that the high GGT activity of cord sera is probably due to hepatic immaturity rather than maternal sources, enzymatic induction or microsomal lesions; that the predominant form of GGT in cord serum may be a complex with HDL and less sialized than the adult enzyme; and that, of the factors examined, the best indicator of neonatal maturity is fetal haemoglobin.

Abnormal serum immunoglobulin concentrations in patients with diabetes mellitus.

Since the recently reported relationship between serum fructosamine and IgA concentrations appears to throw doubt on the clinical utility of fructosamine as a measure of hyperglycemic status if IgA concentration is not taken into account, we studied serum immunoglobulin concentrations in 169 diabetics and their relationship with various clinical and analytical parameters. Over 41% of the patients studied had abnormal serum IgA concentrations. Serum IgA concentration was negatively correlated with serum albumin, and among IDDM patients was positively correlated with age (so that the prevalence of abnormal IgA was 57.7% among IDDM patients aged over 30 years). Among NIDDM patients, abnormal IgA concentrations were especially prevalent among those being treated with oral hypoglycemics. Abnormal IgA was also more frequently found in both IDDM and NIDDM patients, who had been under treatment for 10 years or more. Abnormal IgG concentrations were found in 11.8% of the diabetics, and the mean IgM concentration found in the patients was 41.6% lower than in the normoglycemic group. We conclude that abnormal serum IgA concentrations are very common in diabetic patients and that further research should be carried out to verify whether the determination of serum immunoglobulins, IgA in particular, is of clinical use for monitoring diabetes or evaluating its secondary effects.

Purine and carnitine metabolism in muscle of patients with Duchenne muscular dystrophy.

We determined levels of purines, purine metabolites, related enzymes and carnitine in muscle of 8 untreated Duchenne muscular dystrophy (DMD) patients, 12 allopurinol-treated DMD patients and 12 age-matched controls. Muscle of DMD patients was found to be deficient in ATP, ADP, adenylsuccinate, hypoxanthine, guanine and adenylsuccinate synthetase. In allopurinol-treated DMD patients, mean total adenylate level was only three times less than in controls (versus 14 times less in untreated DMD patients). Mean inosine monophosphate (IMP), adenine, adenosine, inosine, xanthine, guanine, guanosine and uric acid levels were higher in allopurinol-treated patients than in controls, while mean adenylsuccinate levels were higher than in untreated patients. Allopurinol also restored acylcarnitine levels to normal and significantly increased free carnitine levels. These findings strongly support the hypothesis that Duchenne muscular dystrophy involves alterations leading to blockage of the IMP-->purine pathway and that allopurinol treatment favours restoration of purine levels by this route. Furthermore, our results suggest that the observed deficiencies in cell components unrelated to purine metabolism are long-term secondary effects.

Carnitine deficiency associated with anticonvulsant therapy.

Valproic acid therapy is known to be associated with carnitine deficiency in adult as well as young epileptic patients. In a study of the possible existence of such side-effects with other anticonvulsants, 76.5% of adult patients treated with valproate were deficient in serum free carnitine, with acylcarnitine levels significantly higher than in controls (p less than 0.01), while the carnitine deficiency rate in a group of patients treated with anticonvulsants other than valproate was 21.5%. Since in clinical practice only about one fifth of patients are treated with valproate, this means that about 15% of epileptics are carnitine deficient because of valproate treatment and 17% because of other anticonvulsants. The mechanisms and clinical and biological consequences of the carnitine deficiency associated with antiepileptic drugs other than valproate are not known.

Carnitine deficiency in haemodialysed patients.

Free carnitine, acylcarnitine and total carnitine concentrations have been determined in the sera of chronic renal insufficiency patients undergoing regular haemodialysis treatment and in those of healthy controls. The most striking difference was found to be the high proportion of acylated carnitine (23.4 mumol/l) in the haemodialysed patients. Free carnitine and acylcarnitine levels were not completely restored between successive dialysis treatments, making levels measured immediately before the third weekly sessions significantly lower than those measured before the first session (p less than 0.01). In patients monitored throughout 25 wk of treatment, there was an exponential decay of both total serum carnitine levels (Spearman's r = -0.993, p less than 0.001) and free carnitine levels (Spearman's r = -0.972, p less than 0.001). It is suggested that in the absence of exogenous supplies of carnitine, endogenous synthesis is unable to make up for losses due to dialysis treatment, and that carnitine deficiency consequently ensues.

Carnitine concentrations in dialysed and undialysed patients with chronic renal insufficiency.

Free carnitine, acylcarnitine and total carnitine serum concentrations have been measured in chronic renal insufficiency patients under conservative treatment, in patients under regular haemodialysis treatment and in healthy controls. In the undialysed patients the levels of free carnitine, acylcarnitine and total carnitine were all clearly higher than those of the control group. The free carnitine and total carnitine levels of undialysed subjects were also higher than in regularly haemodialysed patients, showing that dialysis produces plasma carnitine losses that are not compensated for by endogenous synthesis of carnitine (this finding supports published reports of tissue carnitine deficiency in patients undergoing regular haemodialysis). The acylcarnitine levels of dialysed and undialysed patients were not significantly different, however; both were very much higher than that of control group. The hypercarnitinaemia of the patients under conservative treatment suggests that the impairment of renal function causes a reduction in the elimination of carnitine via the kidney.

Concentrations of nucleotides, nucleosides, purine bases, oxypurines, uric acid, and neuron-specific enolase in the cerebrospinal fluid of children with sepsis.

To determine the effects of sepsis on cerebral energy metabolism, the cerebrospinal fluid adenosine monophosphate, inosine monophosphate, inosine, adenosine, guanosine, hypoxanthine, xanthine, and urate concentrations were determined by high-performance liquid chromatography and the neuron-specific enolase levels by means of an enzyme immunoassay method in 32 children with sepsis, without meningitis, aged between 2 months and 13 years, and in 160 age-matched controls. The septic group had significantly higher cerebrospinal fluid concentrations of inosine, adenosine, xanthine, and urate than controls. These results suggest that sepsis could provoke some degree of neuronal hypoxia and significant alterations of cerebral energy metabolism homeostasis.

Serum carnitine levels in epileptic children before and during treatment with valproic acid, carbamazepine, and phenobarbital.

Serum levels of free, acyl, and total carnitine were determined in 32 patients with seizures, before and after 3, 6, and 12 months of treatment with valproic acid (17 patients), carbamazepine (10 patients), or phenobarbital (5 patients). In all three treated groups, both free and total carnitine levels showed a significant decline with respect to pretreatment levels. This decline was most marked and most consistent in patients treated with valproic acid. In 35% of the patients in this group, carnitine deficiency (ie, total carnitine < 30 micromol/L) was observed by month 12. In none of the three groups were serum carnitine levels significantly correlated with the serum concentration of the drug. These findings suggest a need to monitor serum carnitine levels in children treated with any of these drugs.

Cerebrospinal fluid purine metabolite and neuron-specific enolase concentrations after febrile seizures.

If febrile seizures cause significant compromise of neuronal metabolism (whether permanent or reversible), this should be reflected in an increase in the cerebrospinal fluid concentrations of neuron-specific enolase (NSE) and/or adenosine triphosphate (ATP) breakdown products. In the present study, AMP, IMP, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine, uric acid and NSE concentrations were determined in the cerebrospinal fluid of 90 children 1 h after febrile seizure (73 simple febrile seizures (SFS); 17 complex febrile seizures (CFS)), and in a control group of 160 children. There was no statistically significant difference between the SFS group and the control group for any of the substances determined, suggesting that SFS neither significantly depletes neuronal ATP concentration, nor significantly increases NSE concentration; thus, SFS do not appear to constitute a threat to neuronal integrity. However, patients with CFS showed significantly lower IMP concentrations and significantly higher adenine concentrations than controls, and significantly higher AMP concentrations than SFS patients; these results suggest that CFS may affect energy metabolism in the brain. However, NSE concentrations were normal in the cerebrospinal fluid of both SFS and CFS patients, suggesting that neither type of seizure causes significant neuronal damage, at least early after the seizure.

Neuron-specific enolase levels in the cerebrospinal fluid of neurologically healthy children.

Levels of neuron-specific enolase (NSE) levels in the cerebrospinal fluid (CSF) of children without neurological disease were assessed. CSF samples were obtained from 37 subjects aged between 1 month and 13 years. All subjects had undergone lumbar puncture for diagnostic purposes, and were subsequently shown not to be suffering any form of neurological disease. NSE levels in CSF were determined by an enzyme immunoassay method. NSE level ranged from below the detection limit to 4.8 ng/ml (1.52+/-1.01 ng/ml). The present results may be useful as a basis for defining reference levels of NSE in CSF in post-neonatal children.

On-line high-performance liquid-chromatographic separation and cold vapor atomic absorption spectrometric determination of methylmercury and inorganic mercury.

A reversed-phase high-performance liquid chromatography (HPLC) method with cold vapor atomic absorption spectrometry (CV-AAS) detection is developed for mercury speciation. In this paper, the efficiency of tetrabutylammonium bromide reagent and sodium chloride in a methanol-water mixture as mobile phase is evaluated for HPLC separation of methylmercury and inorganic mercury coupled with on-line CV-AAS determination. Both mercury species are separated on a reversed-phase C(18) column. Several parameters (e.g. composition and flow-rate of mobile phase) are investigated for the optimization of HPLC separations. CV-AAS technique parameters are also studied for their effect on sensitivity (sodium borohydride and sodium hydroxide concentrations in the reducing agent, reducing agent flow-rate, length of the reduction coil and nitrogen flow-rate). Quantitative recoveries for both inorganic mercury and methylmercury are obtained from a spiked natural water sample.