Jacopo Gervasoni

Folate, vitamin B12 and homocysteine status in an Italian blood donor population

BACKGROUND AND AIMS The relevance of folate, other B-vitamins and homocysteine (Hcy) for the occurrence or prevention of several diseases has induced growing interest. Unfortunately, little evidence is available regarding B-vitamin concentrations in Italy. This study evaluated in a region of middle-southern Italy, folate, vitamin B12 and Hcy concentrations and the prevalence of their ideal blood levels. The main determinants of B-vitamins and Hcy were also considered. METHODS AND RESULTS Male and female blood donors (n=240), aged 18-66 years and living in Molise region (Italy), were enrolled in the study. They completed a brief questionnaire concerning fruit and vegetables intake, physical activity and smoking; serum and red blood cell (RBC) folate and serum vitamin B12 were measured by an immunoassay on an automated analyzer. Total Hcy was measured by high performance liquid chromatography (HPLC). Geometric means of serum folate, RBC folate and serum vitamin B12 were 10.8nmoll(-1), 426.0nmoll(-1) and 245.0pmoll(-1), respectively. Only 22.5%, 24.2% and 16.3% of blood donors showed an adequate level of serum folate, RBC folate or serum vitamin B12 respectively. When a cut-off of RBC folate ≥906nmoll(-1) was used no women of childbearing age had adequate levels. A geometric mean of 14.0μmoll(-1) was found for total Hcy, with an ideal concentration in 12.1% of subjects. Folate concentration was higher in women and non-smokers and in subjects with higher consumption of fruit and vegetable. CONCLUSION This study shows a low-moderate B-vitamins status in middle-southern Italy, associated with an inadequate fruit and vegetable consumption. A public health strategy should be undertaken to encourage a B-vitamin-rich diet with the addition of vitamin supplements or vitamin fortified foods in population subgroups with special needs.

A simple liquid chromatography-tandem mass spectrometry method for urinary free cortisol analysis: suitable for routine purpose.

Abstract Background: The best index of adrenal dysfunction is urinary free cortisol (UFC) measurements performed using a 24-h urine collection. This measurement is also useful in the investigation of Cushings syndrome. In this paper, we report a simple and selective method for the analysis of UFC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) suitable for use in a high-volume clinical laboratory routine. The results were compared to those obtained using a commercial immunoassay method used in our laboratory. Methods: Urine samples containing 50 ng of internal standard (Cortisol-9,11,12,12-d4) were deproteinized using centrifugal filters with a molecular weight 10,000 Da cut-off and injected on a reversed phase column. Cortisol was analyzed in highly selective reaction monitoring in positive atmospheric pressure chemical ionization mode, at a resolution of 0.4 amu full width half maximum, and following the transitions related to the precursor 363.2 for cortisol and 367.2 for deuterated cortisol. The method validation included analysis of precision, linearity, sensitivity, recovery and interference from structurally similar steroids. UFC from 230 subjects was measured using LC-MS/MS and electrochemiluminescence immunoassay (ECLIA) methods. Results: The calibration curves exhibited linearity and reproducibility in the range 7–10,000 nmol/L. Total imprecision was lower than 10%. The limit of detection and limit of quantification were 2 and 7 nmol/L, respectively. Mean recovery was higher than 90%. Structurally similar steroids do not interfere in the proposed method, but cause a significant change in the ECLIA results. Cortisol values obtained using the ECLIA method were always higher than those obtained using the LC-MS/MS method, with the bias directly proportional to cortisol concentrations. The reference values calculated using 180 normal subjects were 11–70 μg/day. Conclusions: The proposed method is sensitive, simple, free from interferences and reliable for routine use. Clin Chem Lab Med 2010;48:1433–7.

A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC-MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80  v/v, containing 0.1% formic acid and 1.5  mmol/L ammonium formate in the water phase) at a flow rate of 0.250  mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.

A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids

Background: Low molecular weight thiols (cysteine, cysteinylglycine, glutathione [GSH] and homocysteine) are important intermediates in different metabolic pathways. Glutathione has a relevant role as an antioxidant in detoxification of toxic compounds and xenobiotics, and homocysteine represents a risk factor for cardiovascular, neurological, and congenital diseases. Homocysteine and GSH are metabolically related in a pathway including cysteine and cysteinylglycine as intermediates. For these reasons, determination of homocysteine and related thiols is of great importance in the diagnosis of several diseases. Methods: The measurement of these sulphur compounds can be performed by using different methods, such as liquid or gas chromatography/mass spectrometry, highperformance liquid chromatography (HPLC), or capillary electrophoresis. Results: This study describes an HPLC method coupled with fluorimetric detection for the simultaneous determination of cysteine, cysteinylglycine, GSH, and homocysteine in different biological fluids (blood, saliva, urine, and cerebrospinal fluid [CSF]). Conclusion: The comparison of the results obtained by other authors, as well as the method validation and the analytical costs, indicate that this HPLC method is particularly suitable for routine measurement of thiols in different body fluids.

Lutein absorption in premature infants.

The objective of this study was to measure lutein and zeaxanthin plasma levels after oral lutein administration in preterm infants. Lutein was given orally in a single dose of 0.5 mg/kg to 10 preterm infants at a mean age of 52 h of life. Plasma lutein and zeaxanthin were measured before and 6, 24, 48, and 120 h after lutein administration. All infants had detectable plasma levels of lutein and zeaxanthin before treatment. Lutein concentration increased by 13.5% at 6 h and by 16.7% at 24 h, and returned to the basal level at 120 h after treatment. Zeaxanthin remained unchanged during the study period. Lutein is well absorbed in preterm infants when given orally. The clinical impact of increasing plasma lutein concentrations on macular development and visual function needs further investigation.

FT-IR Analysis of Urinary Stones: A Helpful Tool for Clinician Comparison with the Chemical Spot Test

Background. Kidney stones are a common illness with multifactorial etiopathogenesis. The determination of crystalline and molecular composition and the quantification of all stone components are important to establish the etiology of stones disease but it is often laborious to obtain using the chemical method. The aim of this paper is to compare chemical spot test with FT-IR spectroscopy, for a possible introduction in our laboratory. Methods. We analyzed 48 calculi using Urinary Calculi Analysis kit in accordance with the manufacturers instructions. The same samples were analyzed by FT-IR using the Perkin Elmer Spectrum One FT-IR Spectrometer. All FT-IR spectra of kidney stones were then computer matched against a library of spectra to generate a report on the various components. Results. On the basis of FT-IR analysis, the 48 calculi were divided into three groups: pure stone, mixed stone, and pure stone with substances in trace. Results of each group were compared with those obtained with chemical spot test. A general disagreement between methods was observed. Conclusions. According to our data, the introduction of the FT-IR technique in clinical chemistry laboratory may be more responsive to clinician expectations.

Effects of lutein supplementation on biological antioxidant status in preterm infants: a randomized clinical trial.

Abstract Objective: To test the hypothesis that lutein, compared to the placebo, would enhance the total antioxidant status (TAS) in the preterm infants. Methods: Infants with gestational age (GA) ≤34 weeks were randomly assigned to receive a daily dose of lutein and zeaxanthin (0.5 mg + 0.02 mg/kg/d) or placebo from the 7th day of life until 40th week of postmenstrual age or until discharge. Results: Seventy-seven preterm infants were randomized (38 in the Lutein group and 39 in the Placebo group) with mean GA of 30.4 (±2.3) weeks and the mean birth weight of 1415 (±457) grams. The TAS did not result statistically different between the two groups during all the study period, but a significant linear correlation was evidenced between plasma lutein concentration and TAS (r = 0.14, p = 0.012) and between plasma zeaxanthin concentration and TAS (r = 0.13, p = 0.02). Conclusions: Supplementation of preterm infants with orally lutein was ineffective in enhancing biological antioxidant capacity. Further studies need to better understand the bioavailability of lutein in the neonatal period in order to identify any best form of supplementation. Trial registration number: UMIN000007041.

Assessment of salivary free cortisol levels by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in patients treated with mitotane.

OBJECTIVEMitotane is an adrenocytolytic agent used in adrenocortical carcinoma, inducing adrenal insufficiency, requiring replacement treatment. Such therapy is not easy to monitor because of mitotane interference. Salivary cortisol reflects a free fraction of plasma cortisol and may be useful in such patients.DESIGNThe aim of our study was to evaluate salivary cortisol by HPLC coupled to tandem-mass spectrometry (LC-MS/MS) and by an electrochemi-luminescence immunoassay (ECLIA) in patients treated with mitotane. We enrolled 6 patients receiving mitotane and 2 Addison disease patients as negative controls and determined salivary cortisol rhythm. We also determined the salivary cortisol rhythm in 8 healthy subjects. Salivary samples (n=112) were assayed by ECLIA, using Roche Modular E170, and by LC-MS/MS.RESULTSThe mean values obtained by ECLIA were significantly higher than those obtained by LC-MS/MS in the mitotane group (p<0.001). In fact, in the group measured by LC-MS/ MS, we observed several peaks eluting at a retention time different from the cortisol group, presumably due to cortisol-like analogues. In Addison disease, since steroidogenesis is absent, salivary cortisol values measured by the two methods did not show any significant difference (p=0.61).CONCLUSIONSSalivary cortisol measured by LC-MS/MS is a selective method, excluding cortisol analogues accumulating in treated patients. Therefore, LC-MS/MS offers an effective system to monitor replacement therapy in mitotane treated patients.

Plasma Thiols Levels in Alzheimer's Disease Mice under Diet-Induced Hyperhomocysteinemia: Effect of S-Adenosylmethionine and Superoxide-Dismutase Supplementation

Widely confirmed reports were published on association between hyperhomocysteinemia, B vitamin deficiency, oxidative stress, and amyloid-β in Alzheimers disease (AD). Homocysteine, cysteine, cysteinylglycine and glutathione are metabolically interrelated thiols that may be potential indicators of health status and disease risk; they all participate in the metabolic pathway of homocysteine. Previous data obtained in one of our laboratories showed that B vitamin deficiency induced exacerbation of AD-like features in TgCRND8 AD mice; these effects were counteracted by S-adenosylmethionine (SAM) supplementation, through the modulation of DNA methylation and antioxidant pathways. Since the cellular response to oxidative stress typically involves alteration in thiols content, a rapid and sensitive HPLC method with fluorescence detection was here used to evaluate the effect of SAM and superoxide-dismutase (SOD) supplementation on thiols level in plasma, in TgCRND8 mice. The quantitative data obtained from HPLC analysis of mice plasma samples showed significant decrease of thiols level when the B vitamin deficient diet was supplemented with SAM + SOD and SOD alone, the latter showing the greatest effect. All these considerations point out the measurement of plasma thiols concentration as a powerful tool of relevance for all clinical purposes involving the evaluation of oxidative stress. The coupling of HPLC with fluorimetric detection, here used, provided a strong method sensitivity allowing thiols determination at very low levels.

Lutein and zeaxanthin concentrations in formula and human milk samples from Italian mothers

Lutein and zeaxanthin concentrations in formula and human milk samples from Italian mothers