UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects.

Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells.

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC's anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Stability of Vitamin A, Iron and Zinc in Fortified Rice during Storage and Its Impact on Future National Standards and Programs--Case Study in Cambodia.

Fortified rice holds great potential for bringing essential micronutrients to a large part of the world population. The present study quantified the losses of three different micronutrients (vitamin A, iron, zinc) in fortified rice that were produced using three different techniques (hot extrusion, cold extrusion, and coating) and stored at two different environments (25 ± 5 °C at a humidity of 60% and 40 ± 5 °C at a humidity of 75%) for up to one year. Fortified rice premix from the different techniques was mixed with normal rice in a 1:100 ratio. Each sample was analyzed in triplicate. The study confirmed the high stability of iron and zinc during storage while the retention of vitamin A was significantly affected by storage and the type of techniques used to make rice premix. Losses for iron and zinc were typically <10% for any type of rice premix. After 12 months at mild conditions (25 °C and humidity of 60%), losses for vitamin A ranged from 20% for cold extrusion, 30% for hot extruded rice 77% for coated rice premix. At higher temperatures and humidity, losses of vitamin A were 40%-50% for extruded premix and 93% for coated premix after 6 months. We conclude that storage does lead to a major loss of vitamin A and question whether rice is a suitable food vehicle to fortify with vitamin A. For Cambodia, fortification of rice with iron and zinc could be an effective strategy to improve the micronutrient status of the population if no other food vehicles are available.

Are the Recommended Dietary Allowances for Vitamins Appropriate for Elderly People?

BACKGROUND: An adequate vitamin intake is essential for a good nutritional status, especially in older women, who are more sensitive to nutritional deficiencies. The American, European and Italian Recommended Dietary Allowances (RDAs) derive mainly from studies on adults, and it is not clear whether they also apply to elderly people. Comparing the RDAs with the actual vitamin intake of a group of healthy older women could help to clarify the real needs of elderly people. OBJECTIVE: Our aim was to compare the American, European, and Italian RDAs with the actual vitamin intake of a group of healthy older women. DESIGN: This was a cross-sectional study. PARTICIPANTS: The study included 286 healthy women aged older than 65 years. MAIN OUTCOME MEASURES: For each micronutrient, the 50th percentile of the distribution of its intake was considered as the average requirement, and the corresponding calculated RDA for our sample was the average requirement×1.2, as recommended by the US Food and Nutrition Board. This calculated RDA was then compared with the American, European, and Italian RDAs. STATISTICAL ANALYSES PERFORMED: Student's t test or the Mann-Whitney test (after checking the normal distribution of the micronutrient) for continuous variables; the χ(2) test for categorical variables. RESULTS: The calculated RDA were 2,230 µg retinol equivalents for vitamin A, 2.8 µg for vitamin B-12, 0.9 mg for thiamin, 1.4 mg for riboflavin, 3.6 mg for pantothenic acid, 1.4 mg for vitamin B-6, 320 µg for folic acid, and 115 mg for vitamin C. CONCLUSIONS: Our findings suggest that the current RDAs are adequate for older women's intake of riboflavin, vitamin B-6, and folic acid, but should be raised for vitamin B-12 and for vitamin C.

Validation study of the VitaKit A test kit for the determination of vitamin A in fluid milk (2% fat) for routine quality control. Performance Tested Method 061001.

A validation study of VitaKit A for quantitation of total vitamin A in 2% fluid milk was carried out according to the guidelines provided by AOAC INTERNATIONAL. The VitaKit A was compared, in terms of repeatability and accuracy, with the U.S. Food and Drug Administration-Interstate Milk Shippers HPLC reference method for determination of total vitamin A in fluid milk with 2% fat. The data obtained by the VitaKit A method are in excellent agreement with the data obtained by the HPLC reference method. Further, a low LOD (0.33 international unit/mL) was obtained for the VitaKit A method; the presence of interferents, like cholesterol and vitamin D3, in the milk had minor influence on the quantitation of total vitamin A by the VitaKit A method. The VitaKit A test kit was found to be stable for 1 year from the date of manufacture when stored at 2-8 degrees C. The method requires 2 h processing time, compared to 1-2 days for the HPLC reference method. The results of this validation study clearly demonstrate that the VitaKit A method is reliable, rapid, and accurate for the quantitation of total vitamin A in fluid milk containing 2% milk fat. An independent study by Q Laboratories Inc., Cincinnati, OH, under the validation guidelines of AOAC INTERNATIONAL, confirmed these findings.

Serum vitamin A and E analysis: comparison of methods between laboratories enrolled in an external quality assurance programme.

AIM: To survey laboratories enrolled in the Royal College of Pathologists of Australasia (RCPA) Chemical Pathology Quality Assurance Programme (QAP) for vitamin A and E testing to determine differences between methods of analysis. METHODS: A detailed questionnaire covering the major aspects of serum vitamin A and E analysis was sent to all participating laboratories in 2007. RESULTS: Thirteen out of the 22 laboratories completed the questionnaire. Methods between laboratories showed a great deal of commonality. All respondents performed a liquid extraction step, which included the addition of an internal standard, followed by high-performance liquid chromatography (C18 columns with predominantly methanol-based mobile phases) with spectrophotometric detection. Major inter-laboratory differences were whether the sample was protected from light, the extraction solvents and ratios used, the drying down temperature used post-liquid extraction and choice of calibrator. CONCLUSIONS: The questionnaire highlighted discrete methodological differences between laboratories. These findings provide direction to enable the Vitamins Working Party of the Australasian Association of Clinical Biochemists to further investigate the dispersion in results between participants of the RCPA QAP vitamin programme.

Efficacy validation of synthesized retinol derivatives In vitro: stability, toxicity, and activity.

Retinol (vitamin A) is used as an antiwrinkle agent in the cosmetics industry. However, its photo-instability makes it unsuitable for use in general cosmetic formulations. To improve the photo-stability of retinol, three derivatives (3, 4, and 5) were synthesized and their biological activities were analyzed. 1H NMR and HPLC analysis indicated that derivatives 3 and 5 were much more stable than retinol under our sunlight exposure conditions. When human adult fibroblasts were treated, the IC(50) of derivative 3 was 96 microM, which is similar to that of retinol, as determined by the MTT assay. Derivatives 4 and 5 were 2.5 and 8 times more toxic than retinol, respectively. At 1 microM treatment, like retinol, derivatives 3 and 4 were specifically active for RARalpha out of six retinoid receptors (RAR/RXRalpha, beta, gamma). Dose-dependent analysis confirmed that derivative 4 was as active as retinol and the other two derivatives were less active for RARalpha. The effect of our derivatives on the expression of collagenase, an indicator of wrinkle formation, was measured using the transient co-expression of c-Jun and RT-PCR in HaCaT cells. Collagenase promoter activity, which is increased by c-Jun expression, was reduced 42% by retinol treatment. The other derivatives inhibited collagenase promoter activity similarly. These results were further confirmed by RT-PCR analysis of the collagenase gene. Taken together, our results suggest that retinol derivative 3 is a promising antiwrinkle agent based on its higher photo-stability, lower RARalpha activity (possibly indicating reduced side effects), and similar effect on collagenase expression.

Vitamin A and sudden infant death syndrome in Scandinavia 1992-1995.

AIM: To assess the effect of vitamin supplementation on the risk of sudden infant death syndrome (SIDS). METHODS: The analyses are based on data from the Nordic Epidemiological SIDS Study, a case-control study in which parents of SIDS victims in the Scandinavian countries were invited to participate together with parents of four matched controls between 1 September 1992 and 31 August 1995. The odds ratios presented are computed by conditional logistic regression analysis. RESULTS: The crude odds ratio in Scandinavia for not giving vitamin substitution was 2.8 (95% CI (1.9, 4.3)). This effect was statistically significant in Norway and Sweden, which use A and D vitamin supplementation, but not in Denmark, where only vitamin D supplementation is given. The odds ratios remained significant in Sweden when an adjustment was made for confounding factors (OR 28.4, 95% CI (4.7, 171.3)). CONCLUSION: We found an association between increased risk of sudden infant death syndrome and infants not being given vitamin supplementation during their first year of life. This was highly significant in Sweden, and the effect is possibly connected with vitamin A deficiency. This effect persisted when an adjustment was made for potential confounders, includingsocioeconomic factors.

Method for the simultaneous determination of retinol and beta-carotene concentrations in human tissues and plasma.

To understand differential tissue distribution of retinoids and carotenoids, as it might influence biological processes in humans, we developed and demonstrated a method for measuring them in selected human tissues. The method includes internal standards and a secondary reference standard to eliminate the need for external standard calibration and to minimize sample-handling errors. Tissues were digested (saponified) in ethanolic KOH. Retinol and beta-carotene were extracted with organic solvent containing internal standards. Analytes were separated using isocratic liquid chromatography and quantified at 325 nm for retinol and 450 nm for beta-carotene. Plasma was analyzed in a similar way but without saponification. Retinal-O-ethyloxime and beta-apo-12'-carotenal-O-t-butyloxime served as internal standards. Plasma, breast, and fat from breast surgery patients and colon, liver, muscle, and fat from colon surgery patients were analyzed. Within-day relative standard deviations (RSDs) for plasma were <0.04 for beta-carotene and <0.03 for retinol, between-day RSDs were <0.05 for beta-carotene and <0.04 for retinol. Saponification ensured complete extraction of retinol and beta-carotene and removal of triglycerides that "foul" chromatographic columns. It seems retinol and beta-carotene concentrations in tissues and blood of cancer patients are the same or higher than those in corresponding tissues of patients without these cancers.

Vitamin A deficiency disorders.

The major cause of blindness in children worldwide is xerophthalmia caused by vitamin A deficiency. In addition it has other adverse effects, including increased mortality and the term vitamin A deficiency disorders (VADD) has been introduced to cover the whole clinical spectrum of disease. The ocular manifestations of xerophthalmia have been classified and a set of prevalence criteria for the detection of a problem of public health magnitude has been in use for more than two decades. The global prevalence of VADD is now well documented and World Health Organisation (WHO) receives information continuously for updating its data base on the subject. The pathogenesis of the disease is still imperfectly understood, it is not at all clear precisely why certain subjects in vulnerable communities develop xerophthalmia, while the majority are spared. A schedule for treatment of the established case has been available for a long time, but at both clinic and hospital level concentrated sources of vitamin A for treatment are frequently not available. More emphasis needs to be laid on prevention and a choice of methods consisting of large dose supplementation, fortification of food, control of precipitating infections and dietary improvement. The advantages and drawbacks of each are discussed.

PIP: This article focuses on xerophthalmia, caused by vitamin A deficiency, which is the major cause of blindness among children worldwide. The term "vitamin A deficiency disorders" (VADD) has been introduced to cover the whole clinical spectrum of disease. The global prevalence of VADD is now well documented and WHO has evidence that the number of young children suffering from subclinical vitamin A deficiency is steadily increasing. Moreover, the pathogenesis of the disease is still imperfectly understood. It is not clear why certain subjects in vulnerable communities develop xerophthalmia, while the majority are spared. Oral preparations of vitamin A are well absorbed and utilized in even severely malnourished patients. A schedule of treatment is given to serve as a guide in the full treatment process. Control of VADD involves a massive dose supplementation, fortification of common foodstuffs with vitamin A, immunization, and dietary improvement. However, each of these methods has drawbacks and advantages.

[Retinol Acetate Reference Standard (Control 971) and Retinol Palmitate Reference Standard (Control 971) of National Institute of Health Sciences].

The "Retinol Acetate Reference Standard (Control 971)" and "Retinol Palmitate Reference Standard (Control 971)" of National Institute of Health Sciences using the assay of vitamin A ester were prepared. The proposed materials were evaluated in collaboration with four laboratories. Analytical data obtained were as follows. 1) The purities of retinol acetate and retinol palmitate measured by HPLC were 99.9 +/- 0.06% and 94.5 +/- 0.06%, respectively. 2) ultraviolet spectrum of retinol acetate and retinol palmitate showed the lambda max at 326-327 nm. 3) The difference in relative extinction of retinol acetate and retinol palmitate at 300 nm, 310 nm, 320 nm, 330 nm, 340 nm and 350 nm are within the range provided in JPXIII. 4) The contents of retinol acetate and retinol palmitate were 52,000 IU/g and 52,200 IU/g, respectively. Based on the above results, these proposed materials were authorized to be the Reference Standards of the National Institute of Health Sciences.

Decreasing traditional food use affects diet quality for adult Dene/Métis in 16 communities of the Canadian Northwest Territories.

We assessed diets in 16 Dene/Métis communities in the Canadian Arctic. We described nutrient intakes and identified nutrients at risk among adult Dene/Métis, evaluated the influence of traditional food on diet quality, and examined the direction of dietary change by comparing intergenerational and between-community differences in dietary intake. Diet varied according to sex, age and community. Nutrients of possibly inadequate intake (irrespective of subject sex, age or community) included calcium, vitamin A and folic acid. Dietary fiber intake was also of concern. Traditional food (animals and plants harvested from the local environment) was consumed on 65. 4% of interview days; on those days intakes of iron, zinc and potassium were higher (P < 0.05) and those of sodium, fat, saturated fat and sucrose were lower (P < 0.05) than on days when market food only was consumed. In this population, the shift away from traditional food towards a diet composed exclusively of market food was characterized by an increase (P < 0.05) in absolute energy intake and an increase (P < 0.01) in the relative contributions of carbohydrate (particularly sucrose), fat and saturated fat. This pattern of change calls for initiatives to document the current health status of this population and to prevent potential negative health consequences of dietary change.

Evaluation of multiple micronutrient supplementation in the management of oral submucous fibrosis in Karachi, Pakistan.

Oral submucous fibrosis (OSF) is an oral precancerous condition characterized by symptoms such as intolerance to spicy food, altered salivation, progressive difficulty in opening the mouth, and signs like vesiculation, ulceration, blanching, rigidity, and stiffening of the oral mucosa and depapillation and altered mobility of the tongue. It is seen mostly among people of Indian subcontinent origin. The major structural change is extensive fibroelastic scarring of the lamina propria and deeper connective tissues. A combination of micronutrients (vitamins A, B complex, C, D, and E) and minerals (iron, calcium, copper, zinc, magnesium, and others) was evaluated for its efficacy in controlling the symptoms and signs of OSF in 117 compliant subjects in Karachi, Pakistan, in a single-arm preliminary study. The subjects received supplementation for one to three years. Significant improvement in symptoms, notably intolerance to spicy food, burning sensation, and mouth opening, was observed at exit. The interincisor distance deteriorated in 11 subjects (10%) at exit; it was stable in 56 subjects (49%) and improved in 48 (41%). The mean interincisor distance was 19.1 +/- 10.8 (SD) mm at exit compared with 16.2 +/- 7.5 mm at baseline. A significant proportion of concomitant lesions like leukoplakia also regressed at exit. The major outcome from this study was a beneficial clinical response in subjects with OSF to multiple micronutrient intervention, which justifies its further evaluation in well-designed randomized controlled trials in other settings in South Asia.

Mortality of infants < 6 mo of age supplemented with vitamin A: a randomized, double-masked trial in Nepal.

The effect of supplementing 11,918 infants < 1 mo and 1-5 mo of age with vitamin A (15,000 and 30,000 micrograms retinol equivalents or 50,000 and 100,000 IU, respectively) or a placebo on subsequent 4-mo mortality was assessed in a randomized, double-masked community trial in the rural plains of Nepal. There were 130 deaths (51.6/1000 child-y) in the control group and 150 deaths (57.1/1000 child-y) in the vitamin A group, yielding a relative risk of 1.11 (95% CI: 0.86, 1.42), which is indicative of no overall effect on early infant mortality. There was a tendency for the relative risk of mortality among vitamin A recipients to rise with improved nutritional status. These results suggest that distribution of a large oral dose of vitamin A to infants < 5-6 mo of age may not benefit short-term survival. This is in contrast with the results of trials in which older infants and children in this same population were supplemented.

Dietary vitamin A intake and the incidence of diarrhea and respiratory infection among Sudanese children.

The relationship of vitamin A deficiency and child survival has been documented in a number of studies but not in others, yet the relationship of vitamin A with child morbidity remains controversial. We prospectively examined the relationship of dietary vitamin A intake and the incidences of diarrhea and respiratory infection among 28,753 Sudanese children between the ages of 6 mo and 6 y. Total dietary vitamin A intake was strongly and inversely associated with the risk of diarrhea (multivariate risk in top relative to bottom quintile = 0.58, 95% confidence interval = 0.47-0.72); we also observed a strong inverse association with the risk of having cough and fever (0.60, 0.45-0.81). On the other hand, we noted a significantly positive association of dietary vitamin A intake and incidence of cough alone (1.69, 1.52-1.88), a sign that may be assocsated with a healthy respiratory epithelium. Vitamin A intake was also negatively associated with the risk of measles. These prospective data emphasize the importance of adequate dietary vitamin A intake to protect the health of children in developing countries.

Evaluation of chemoprevention of oral cancer with Spirulina fusiformis.

The blue-green microalgae Spirulina, used in daily diets of natives in Africa and America, have been found to be a rich natural source of proteins, carotenoids, and other micronutrients. Experimental studies in animal models have demonstrated an inhibitory effect of Spirulina algae on oral carcinogenesis. Studies among preschool children in India have demonstrated Spirulina fusiformis (SF) to be an effective source of dietary vitamin A. We evaluated the chemopreventive activity of SF (1 g/day for 12 mos) in reversing oral leukoplakia in pan tobacco chewers in Kerala, India. Complete regression of lesions was observed in 20 of 44 (45%) evaluable subjects supplemented with SF, as opposed to 3 of 43 (7%) in the placebo arm (p < 0.0001). When stratified by type of leukoplakia, the response was more pronounced in homogeneous lesions: complete regression was seen in 16 of 28 (57%) subjects with homogeneous leukoplakia, 2 of 8 with erythroplakia, 2 of 4 with verrucous leukoplakia, and 0 of 4 with ulcerated and nodular lesions. Within one year of discontinuing supplements, 9 of 20 (45%) complete responders with SF developed recurrent lesions. Supplementation with SF did not result in increased serum concentration of retinol or beta-carotene, nor was it associated with toxicity. This is the first human study evaluating the chemopreventive potential of SF. More studies in different settings and different populations are needed for further evaluation.

Analysis of milk-based infant formula. Phase V. Vitamins A and E, folic acid, and pantothenic acid: Food and Drug Administration-Infant Formula Council: collaborative study.

In 1982, the U.S. Food and Drug Administration, the Infant Formula Council and its member companies, contract laboratories, and other government laboratories began a study of analytical methods for the nutrients listed in the Infant Formula Act of 1980 (P.L. 96-359). Four phases of the study have been completed and are discussed in earlier reports. The present report provides data on Phase V, in which 13 laboratories collaboratively studied individual methods for folic acid, pantothenic acid, and vitamin E, in addition to 2 methods for vitamin A. Vitamins A and E are determined by liquid chromatography. Folic acid and pantothenic acid are determined by microbiological methods using acidimetric and/or turbidimetric assays as the determinative step. In most cases, relative standard deviations for repeatability, RSDr, and reproducibility, RSDR, are as good as those that would be predicted from other collaborative studies. RSDr and RSDR values obtained for the 5 methods are 9.35 and 25.44% for folic acid, 4.59 and 10.23% for pantothenic acid, 8.46 and 11.69% for vitamin E, 3.62 and 9.72% for vitamin A (retinol isomers), and 4.9 and 10.5% for vitamin A (retinol). The 5 methods have been adopted first action by AOAC International.