Elio F. De Palo

Cortisol assays and diagnostic laboratory procedures in human biological fluids

The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.

Separation methods for acyclovir and related antiviral compounds.

Acyclovir (ACV) is an antiviral drug, which selectively inhibits replication of members of the herpes group of DNA viruses with low cell toxicity. Valaciclovir (VACV), a prodrug of ACV is usually preferred in the oral treatment of viral infections, mainly herpes simplex virus (HSV). Also other analogues such as ganciclovir and penciclovir are discussed here. The former acts against cytomegalovirus (CMV) in general and the latter against CMV retinitis. The action mechanism of these antiviral drugs is presented briefly here, mainly via phosphorylation and inhibition of the viral DNA polymerase. The therapeutic use and the pharmacokinetics are also outlined. The measurement of the concentration of acyclovir and related compounds in biological samples poses a particularly significant challenge because these drugs tend to be structurally similar to endogenous substances. The analysis requires the use of highly selective analytical techniques and chromatography methods are a first choice to determine drug content in pharmaceuticals and to measure them in body fluids. Chromatography can be considered the procedure of choice for the bio-analysis of this class of antiviral compounds, as this methodology is characterised by good specificity and accuracy and it is particularly useful when metabolites need to be monitored. Among chromatographic techniques, the reversed-phase (RP) HPLC is widely used for the analysis. C18 Silica columns from 7.5 to 30 cm in length are used, the separation is carried out mainly at room temperature and less than 10 min is sufficient for the analysis at 1.0-1.5 ml/min of flow-rate. The separation methods require an isocratic system, and various authors have proposed a variety of mobile phases. The detection requires absorbance or fluorescence measurements carried out at 250-254 nm and at lambdaex=260-285 nm, lambdaem=375-380 nm, respectively. The detection limit is about 0.3-10 ng/ml but the most important aspect is related to the sample treatment, mainly when body fluids are under examination. The plasma samples obtained from human blood are pre-treated with an acid or acetonitrile deproteinization and the supernatant after centrifugation is successively extracted before RP-HPLC injection. Capillary Electrophoresis methods are also discussed. This new analytical approach might be the expected evolution, in fact the analyses are improved with regard to time and performance, in particular coated capillary as well as addition of stabilisers have been employed. The time of analysis is shortened arriving at less than half a minute. Furthermore by using an electrochemical detection, and having a calibration linearity in the range of 0.2-20.0 ng/ml, the detection limit is 0.15 microg/ml. The measurements of acyclovir and penciclovir have been presented but in the future other related drugs will probably be available using CE methods.

Plasma and urine free L-Carnitine in human diabetes mellitus

SummaryL-carnitine is essential for the transport of long-chain fatty acids into mitochondria and their oxidation. Recently, a relationship between plasma free fatty acids (FFA) and L-carnitine metabolism has been observed. Plasma free L-carnitine (FC), FFA, triglycerides, cholesterol, blood glucose concentration and daily excretion of FC were determined in 20 diabetic patients as well as in 18 control subjects. Both in male diabetics and in male controls, plasma FC was significantly higher than in females. Mean plasma FC was found to be significantly reduced in diabetics (21 ± 2vs 35 ± 2 Μmol/1 in non-diabetic subjects; p<0.005). Daily urinary excretion of FC was clearly lower in diabetic patients than in controls (172 ± 34vs 403 ± 38 Μmol/24 h; p<0.001). The reduced plasma FC in diabetes mellitus may be due to redistribution between circulating free and esterified carnitine and to increased utilization of FC for synthesis of acylcarnitine in tissues.

GH/IGF system, cirrhosis and liver transplantation

The GH-related effects are primarily mediated by insulin-like growth factor I (IGF-I), a peptide hormone almost completely produced by the liver. Liver cirrhosis is usually accompanied by a fall in protein turnover. Furthermore, an important consequence of chronic liver disease (CLD) is growth hormone/insulin-like growth factor (GH/IGF) axis modification and growth failure. Nutritional status also suffers in this condition, and IGF-I has been proposed as a marker of hepatocellular dysfunction, malnutrition and survival. CLD is characterised by alterations of various clinical biochemistry laboratory parameters. Aminotransferases, bilirubin, plasma proteins, together with prothrombin time and gamma globulins, are usually examined for laboratory diagnostic and/or monitoring purposes. These traditional parameters are also used in the perioperative liver transplantation, but an early signal of graft functioning has still not been established. The aim of the present work is a review of the possibility offered by the clinical biochemistry laboratory GH/IGF investigation in the outcome of liver transplantation.

Human saliva cortisone and cortisol simultaneous analysis using reverse phase HPLC technique

BACKGROUND Hyper-hypo tension (like Cushings syndrome, apparent mineralocorticoid excess syndrome and Addisons disease) diagnostic laboratory requires cortisol (F) analysis. The simultaneous analysis of human saliva F and cortisone (E), the inactive F metabolite, by solid phase extraction and RP-HPLC was studied. METHODS Saliva/standard samples were C18-SPE extracted, dried and resuspended. E and F were analysed by isocratic RP-HPLC (acetonitrile/water 27/73%) and UV detection. In the morning and in the evening Salivette stimulated saliva specimens were collected from healthy volunteers. RESULTS The E and F calibration curve ranges were 11.0-110.0 and 5.5-55.0 nmol/l respectively. The LOD was 0.2 and 0.1 nmol/l for E and F respectively. The intra and inter assay CVs were respectively 2.7-6.6 and 5.6-7.0% for E and 5.8-7.0 and 11.7-13.1% for F. The E and F spiked saliva sample recovery was 99% and 88% respectively. Saliva specimen stability was validated. E and F saliva levels in healthy volunteers were significantly (p<0.001) higher at 8 a.m. compared with 11 p.m. (26.4+/-8.9 vs. 4.3+/-2.9 nmol/l for E; 11.1+/-4.0 vs. 2.5+/-1.5 nmol/l for F, respectively). CONCLUSIONS This method is suitable for periodic analyses in a clinical biochemistry laboratory for endocrinology investigation purposes, simultaneously analysing E and F levels in a saliva specimen.

Growth hormone/insulin-like growth factor 1 axis recovery after liver transplantation: A preliminary prospective study

Many studies on cirrhotic patients have shown that insulin‐like growth factor 1 (IGF‐1) plasma levels are related to the severity of liver dysfunction. This result suggests that IGF‐1 is probably useful for monitoring liver function in the perioperative course of orthotopic liver transplantation (OLT). Growth hormone (GH), IGF‐1 plasma levels, and routine liver function tests were measured in 15 adult cirrhotic patients undergoing OLT. Measurements were made at the beginning of the operation; during OLT; 24 hours after reperfusion; and in the morning on days 7, 30, and 90. Twenty age‐matched healthy volunteers with normal liver function served as controls. The study group had significantly higher GH levels and lower IGF‐1 levels in the preoperative period compared with the controls. All patients achieved a complete functional hepatic recovery 1 month after OLT, although in 6 of them, the graft had an initial poor function (Group‐IPF). GH and IGF‐1 levels achieved near normal range within 1 week after OLT, and they had no significant correlations with other routine biochemistry tests in this period. IGF‐1 levels in Group‐IPF rose more slowly than in the group with a normal recovery of graft function. Surprisingly, 24 hours after reperfusion, IGF‐1 levels were higher in Group‐IPF than in the group with normal graft function. In conclusion, the severe GH/IGF‐1 axis impairment found in patients with end‐stage cirrhosis reverted very rapidly in the first days after successful OLT. Such a quick, postoperative modulation of IGF‐1 plasma level by the graft suggests that this hormone has the potential to become one of the early indicators of post‐OLT liver function recovery. (Liver Transpl 2004;10:692–698.)

Plasma atrial natriuretic peptide (ANP) fragments proANP (1–30) and proANP (31–67) measurements in chronic heart failure: a useful index for heart trasplantation?

The family of the atrial natriuretic peptides, proANP fragments and the active αANP, is strongly related to heart disease. The aim was to study in CHF subjects the relation of mdANP and NtANP with brain natriuretic peptide (BNP) and with other traditional medical parameters. Sixteen CHF patients (aged 51.9±13.7 years) and 16 healthy subjects age matched (50.8±5.9 years) were selected. Both NtANP and mdANP were higher in CHF patients than in healthy subjects (1436±288 vs. 288±22 pmol/l p<0.001 and 2305±383 vs. 423±65 pmol/l p<0.0001, respectively). BNP in CHF patients was 28.0±9 pmol/l (reference values 1.7±1.8 pmol/l). Both NtANP and mdANP demonstrated positive correlation with BNP, p<0.0001 and with left atrial end-systolic volume, p<0.05. BNP correlated with left ventricular mass, p<0.03. In conclusion, plasma NtANP and mdANP analyses are useful laboratory markers in CHF patient investigation and follow up. In particular, they could be employed as non-invasive parameters to follow up worsening of systolic dysfunction until heart transplantation is required.

Plasma kallikrein activity in human diabetes mellitus

It has recently been observed that administration of bradykinin to diabetic patients improves peripheral glucose utilization. To verify whether there is an alteration of the kallikrein-kinin system in human diabetes, plasma kallikrein activity was measured in 47 diabetic patients and in 20 control subjects. In diabetics plasma kallikrein activity was significantly higher than in controls: 1.04 +/- 0.04 U/ml (p less than 0.001). Although they do not refute the hypothesis that there is an alteration of the kallikrein-kinin system in diabetes mellitus, these findings do not support such a hypothesis either. Increased synthesis of plasma kallikrein activity may be due to increased synthesis of carbohydrate-protein compounds in diabetes mellitus.

Metabolic and endocrine responses to a standard mixed meal. A physiologic study

SummaryIn order to study endocrine and metabolic responses to normal food ingestion, 8 ‘healthy’ subjects received a standard mixed meal which reflected the composition of Western diet (CHO 47%, protein 23%, fat 26%, alcohol 4%), in 20 min. Before and after the meal, in each subject glucose, lactate, FFA, insulin, C-peptide, glucagon and HGH were determined. The results showed that glycemic and insulinemic responses were not very different from those observed after the classical oral glucose tolerance test. Plasma FFA and blood lactate decreased progressively after the meal. Plasma glucagon and HGH showed opposite changes: pancreatic glucagon rose and HGH slightly declined after composite food ingestion.

Physiological and clinical implications of proANP(1-98) circulating levels in the perioperative phase of liver transplantation.

BACKGROUND ProANP(1-126), the prohormone synthesized and secreted by atrial myocites, generates an ANP peptide family, the main forms of which are proANP(1-30), proANP(31-67), proANP(1-98) and proANP(99-126). These molecular circulating forms are involved in hemodynamic and electrolyte homeostasis. In cirrhotic patients, volume homeostasis is almost impaired due to abnormal sodium retention, which results in ascites formation and hemodynamic changes, including high cardiac output and low systemic vascular resistance. During liver transplantation, in the anhepatic phase, hemodynamic instability may occur because of decreased venous return due to surgical manipulation of inferior vena cava, considerable blood loss or cross-clamping. Moreover, marked hemodynamic instability is often observed at the reperfusion of the graft. AIMS The aims of present study are to investigate the changes of ANP during the perioperative phases of Orthotopic Liver Transplantation (OLTx) in end-stage cirrhotic patients. PATIENTS AND METHODS From July to September 1999, 11 cirrhotic patients undergoing to OLTx were included in the study: seven males and four females (average age 46+/-10.4 years) affected by post-alcoholic cirrhosis [Hypertension 15 (1990) 9], post-hepatitis cirrhosis [D.G. Gardner, M.C. Lapointe, B. Kovacic-Milivojevic, C.F. Deschepper, Molecular analisys and regulation of the atrial natriuretic factor gene, in: A.D. Struphers (Ed.), Frontiers in Farmacology and Therapeutics: Atrial Natriuretic Factor, Blackwell, Oxford, England, 1991, pp. 1-22], Wilson disease [Life Sci. 28 (1981) 89] and polycystic disease [Life Sci. 28 (1981) 89], autoimmune cirrhosis [Life Sci. 28 (1981) 89]. In each patient, a hemodynamic assessment was achieved using a Swan-Ganz catheter. Periferical venous samples were performed during and immediately after OLTx for the determination of ANP(1-98) and other biohumoral parameters. RESULTS Mean ANP(1-98) (pmol/ml mean+/-SD) basal levels resulted higher than that recorded in the group of healthy subjects. A significant correlation between 24-h post-reperfusion ANP and intra-operative RBC and RIS requirement was found (p<0.05). The basal values resulted significantly higher than that observed at phase II degrees (p<0.04) and lower than that at phase VI degrees (p<0.05); the anesthetic induction values were significantly lower than that observed at phase VI degrees (p<0.03). CONCLUSIONS ANP(1-98) values may represent a useful marker of hemodynamic derangements during and after OLTx. Further clinical correlations will need a larger patient basis.