Comparison of Tandem Mass Spectrometry and the Fluorometric Method-Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria.

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland-Altman analysis indicated a bias of -38.9% (-23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM.

Introduction of a spectrophotometric method for salivary iodine determination on microplate based on Sandell-Kolthoff reaction.

BACKGROUND: Iodine is an essential element for the synthesis of thyroid hormones. Therefore, a reliable marker of iodine supply is important. Iodine is predominantly excreted via kidneys, but also via salivary glands. Our aim was to introduce a new and simple method for determination of salivary iodine concentration (SLIC). MATERIALS AND METHODS: Self-prepared chemicals and standards for Sandell-Kolthoff reaction on microplate with ammonium peroxydisulfate (AP) in the range 0-400 µg/L were used. Suitability of water-based standards (WBS) and artificial saliva-based standards (ASS) for standard curve were tested. We followed standards for method validation, defined concentration of used AP and compared our results with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). RESULTS: WBS gave more reliable results than ASS as an underestimation of iodine concentration was found for ASS. LoB was 6.5 µg/L, LoD 12.0 µg/L, therefore analytical range was 12-400 µg/L. Intra- and inter-assay imprecisions at iodine concentrations, namely 20, 100, 165, and 350 µg/L were 18.4, 5.1, 5.7, and 2.8%, respectively, and 20.7, 6.7, 5.1, and 4.3%, respectively. Suitable molarity of AP was 1.0 mol/L and showed no difference to 1.5 mol/L (P values for samples with concentration 40, 100, and 150 µg/L, were 0.761, 0.085, and 0.275, respectively), whereas there was a significant change using 0.5 mol/L (P<0.001). Saliva samples could be diluted up to 1:8. There was no interference of thiocyanate and caffeine up to 193.5 mg/L. Our original method was comparable to ICP-MS. Spaerman coefficient was 0.989 (95% CI: 0.984-0.993). CONCLUSIONS: The new method for SLIC determination is in excellent agreement with ICP-MS and easy-to-use.

Interpreting urinary iodine concentration: effects of urine dilution and collection timing.

OBJECTIVES: In population studies, iodine intake estimation relies on median urinary iodine concentration (UIC). However, interpreting UIC measurements can be challenging. METHODS: In our study, we included 772 adult participants from three groups: nationally representative gender-mixed, women of reproductive age, and pregnant women. We measured UIC and urinary creatinine (U-Cr) to calculate the iodine-to-creatinine ratio (I/Cr). U-Cr cut-off value of 0.226 g/L was used for differentiation between diluted and undiluted urine samples. After excluding samples below this cut-off, new median UIC and I/Cr ratios were calculated. We additionally evaluated the influence of urine sample collection time on UIC. RESULTS: Median UICs were 91.8 µg/L for nationally representative group, 58.3 µg/L for women of reproductive age, and 74.9 µg/L for pregnant women, while I/Cr ratios were 91.7, 102.0, and 159.2 µg/g, respectively. After implementing U-Cr cut-off and excluding all data where U-Cr was below cut-off, new median values were 93.4, 76.3, and 95.4 µg/L for UICs, and 88.6, 88.8, and 128.7 µg/g for I/Cr ratios, respectively. In women of reproductive age, median UIC was significantly lower in urine samples collected after 9:30 and after 12:00 as compared to samples collected before 9.30 (53.4, 57.8, and 97.3 µg/L, respectively). CONCLUSIONS: UIC results should be interpreted with caution, considering urine dilution and sample collection timing. U-Cr measurement should be included in population-based iodine intake studies, with corrections applied especially for pregnant women and younger adults, for whom morning is best for single-spot samples.

Diagnostics of Metabolic Bone Disease in Extremely Preterm Infants-Clinical Applicability of Bone Turnover Biochemical Markers and Quantitative Ultrasound.

BACKGROUND: Significant improvement in neonatal care has enabled increasing survival of preterm infants. Metabolic bone disease of prematurity is often overlooked due to other comorbidities of preterm birth. The best approach is screening and prevention of the disease in high-risk infants such as preterm infants. AIM: We followed up the clinical, radiological, and serum biochemical markers of metabolic bone disease in extremely preterm infants (<28 weeks of gestation). The clinical applicability and validation of C-terminal telopeptide of type I collagen (CTX-I) as a novel bone turnover marker were assessed. Standard and novel biochemical bone turnover markers and quantitative ultrasound were compared. METHOD: Patients' data were collected from medical records. Assessments of calcium, phosphate, alkaline phosphatase, bone-alkaline phosphatase, CTX-I, and quantitative ultrasound were prospectively performed twice in 42 extremely preterm infants at postmenstrual ages of 30-32 weeks and 36-40 weeks. Bone mineral density was measured by quantitative ultrasound. CONCLUSION: Phosphate, alkaline phosphatase, bone alkaline phosphatase, calcium, or ionized calcium are not related to gestational age, but bone mineral density, measured by quantitative ultrasound, is related. There is no correlation between standard and novel biochemical markers and quantitative ultrasound for the identification of metabolic bone disease.

Validation of a Spectrophotometric Method for Urinary Iodine Determination on Microplate Based on Sandell-Kolthoff Reaction.

OBJECTIVE: Iodine is an essential part of the thyroid hormones thyroxine and triiodothyronine. Therefore, it is essential to monitor iodine supply in a population. The biochemical marker for assessing and controlling iodine is urinary iodine concentration (UIC). MATERIALS AND METHODS: This cross-sectional study included 180 pregnant women and 308 women of reproductive age. Urine specimens from 185 of the 488 volunteers were used. The urine specimens were measured using 2 methods: (1) ammonium persulfate digestion (APD), followed by the Sandell-Kolthoff (S-K) reaction modified on microplate for spectrophotometric detection; and (2) the reference method, inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The regression equation between the methods was ICP-MS method = 1.137*(APD S-K)-5.57. A Passing-Bablok regression showed no deviation from linearity (P = .17). A Bland-Altman plot showed a negative mean bias of -2.7%. CONCLUSION: The APD S-K reaction modified on microplate for spectrophotometric detection of UIC can be implemented into routine work. Its results are comparable to those of laboratories worldwide and to ICP-MS.

Dietary Intake and Status of Vitamin B12 in Slovenian Population.

Vitamin B12 deficiency poses a health concern, especially in vulnerable populations. Dietary vitamin B12 intake was obtained by two 24 h dietary recalls and food propensity questionnaires in a representative Slovenian cross-sectional food consumption survey, SI.Menu (n = 1248 subjects; 10-74 years). For a subgroup of 280 participants, data on serum vitamin B12 were available through the Nutrihealth study. The estimated usual population-weighted mean daily vitamin B12 intakes were 6.2 µg (adults), 5.4 µg (adolescents), and 5.0 µg (elderly). Lower intakes were observed in females. Inadequate daily vitamin B12 intake (<4 µg) was detected in 37.3% of adolescents, 31.7% of adults, and 58.3% elderlies. The significant predictors for inadequate daily vitamin B12 intake were physical activity score in all age groups, sex in adolescents and adults, financial status and smoking in elderly, and employment in adults. Meat (products), followed by milk (products), made the highest vitamin B12 contribution in all age groups. In adolescents, another important vitamin B12 contributor was cereals. The mean population-weighted serum vitamin B12 levels were 322.1 pmol/L (adults) and 287.3 pmol/L (elderly). Low serum vitamin B12 concentration (<148 nmol/L) and high serum homocysteine (>15 µmol/L) were used as criteria for vitamin B12 deficiency. The highest deficiency prevalence was found in elderlies (7.0%), particularly in males (7.9%). Factors associated with high serum homocysteine were also investigated. In conclusion, although vitamin B12 status was generally not critical, additional attention should be focused particularly to the elderly.

Dietary Iron Intake and Biomarkers of Iron Status in Slovenian Population: Results of SI.Menu/Nutrihealth Study.

Inadequate iron intake and iron deficiency are recognised as a public health problem in the population at large, and particularly in specific subpopulations. Dietary iron intake was analysed using data of the national Slovenian food consumption study, SI.Menu (n = 1248 subjects; 10−74 years), while iron status was evaluated with laboratory analyses of blood haemoglobin, serum ferritin, and iron concentration in samples, collected in the Nutrihealth study (n = 280, adults). The estimated daily usual population-weighted mean iron intakes ranged from 16.0 mg in adults and the elderly to 16.7 in adolescents, and were lower in females for all three age groups. The main dietary iron sources in all the age groups were bread and bakery products, meat (products), fruit, and vegetables. The highest prevalence of haemoglobin anaemia was observed in females aged 51−64 years (6.7%). Critically depleted iron stores (ferritin concentration < 15 µg/L) were particularly found in premenopausal females (10.1%). Factors influencing low haemoglobin, ferritin, and iron intake were also investigated. We observed significant correlations between iron status with meat and fish intake, and with iron intake from meat and fish, but not with total iron intake. We can conclude that particularly premenopausal females are the most fragile population in terms of inadequate iron intake and iron deficiency, which should be considered in future research and public health strategies.

Optimizing the Phenylalanine Cut-Off Value in a Newborn Screening Program.

Phenylketonuria (PKU) was the first disorder for which newborn screening (NBS) was introduced in the early 1960s. Slovenia started the NBS program for PKU in 1979, and the fluorimetric method was implemented in 1992, with a phenylalanine (Phe) cut-off set at 120 mol/L. This value has been in use for almost thirty years and has never been revised. We aimed to analyze the DBS samples and review the data from a large nationwide cohort of newborns to optimize the cut-off values for HFA screening to minimize the number of false positives while maintaining the highest level of sensitivity by detecting all those who needed to be treated. In the first prospective part of the study, we analyzed samples of all newborns in Slovenia in 2019 and 2020, and in the second retrospective part, we reviewed data from all known patients with hyperphenylalaninemia (HFA) in Slovenia born from 2000 to 2018. We defined true screening-positive cases as those that required a low-Phe diet. The sensitivity, specificity and positive predictive values of the modeling elevation of the Phe cut-off value from 120 µmol/L to 200 µmol/L were assessed. The number of recalls at the cut-off of 120 µmol/L was 108 out of 37,784 samples at NBS (2019-2020). Six newborns were defined as true positives and 102 samples as false positives. If the cut-off value was adjusted to 160 µmol/L, only 12 samples exceeded it and all six true positive newborns would be detected. Among the 360,000 samples collected at the NBS between 2000 and 2018, 72 HFA patients in need of a low-Phe diet were found. All the diagnosed cases would have been detected if the cut-off was set to 160 µmol/L. We demonstrated in a large group of newborns (400,000 in 20 years) that using the fluorimetric approach, a cut-off value of 160 µmol/L, rather than 120 mol/L, is safe and that there were no missing true positive patients who required treatment. By increasing the cut-off, this method becomes more precise, resulting in a significantly reduced rate of false positives and thus being less burdensome on both families and the healthcare system.

First Estimation of Reference Intervals for Thyroid-Stimulating Hormone and Thyroid Hormones in Slovenian Population.

For thyroid function estimation and clinical decision making, use of appropriate reference intervals for thyroid-stimulating hormone (TSH), free thyroxine (fT4) and free triiodothyronine (fT3) is crucial. For each laboratory, establishment of own reference intervals is advised. For the first Slovenian estimation of reference intervals for thyroid hormones a large group of 1722 healthy individuals without thyroid disease was established retrospectively. Hormone analyses were performed on automated analyser Advia Centaur XP Immunoassay System (Siemens Healthineers), which reference intervals for TSH, fT4 and fT3 were 0.55-4.78 mIU/L, 11.5-22.7 pmol/L, and 3.5-6.5 pmol/L, respectively. Statistical analysis followed non-parametric percentile method. Our laboratory reference intervals for TSH, fT4 and fT3 are mostly narrower than intervals given by manufacturer. Median value, lower and upper limit for TSH, fT4 and fT3 were 1.98 (0.59-4.23) mIU/L, 14.5 (11.3-18.8) pmol/L and 4.82 (3.79-6.05) pmol/L, respectively. Most likely, an inclusion of a high number of healthy individuals without thyroid disease was a reason for such results.

Dietary Intake of Folate and Assessment of the Folate Deficiency Prevalence in Slovenia Using Serum Biomarkers.

Folate deficiency is associated with various health issues, including anemia, cardiovascular disease, and birth defects. Low folate intake and suboptimal folate status were found in several countries; however, this topic has not yet been investigated in Slovenia. Dietary folate intake and serum folate status were investigated through the nationally representative food consumption study SI.Menu/Nutrihealth. Folate intake was estimated using a sample of N = 1248 subjects aged 10-74 years, stratified in three age groups (adolescents, adults, elderly population), through two 24 h-dietary recalls and food propensity questionnaire. Data on serum folate and homocysteine was available for 280 participants. Very low folate intake (<300 µg/day) was observed in 59% of adolescents, 58% of adults and 68% of elderlies, and only about 12% achieved the WHO recommended level of 400 µg/day. Major dietary contributors were vegetables and fruit, and cereal products. Living environment, education, employment status and BMI were linked with low folate intake in adults; BMI, and sex in adolescents; and sex in elderlies. Considering low serum folate (<7 nmol/L) and high serum homocysteine (>15 nmol/L), folate deficiency was found in 7.6 and 10.5% in adults and elderlies, respectively. Additional public health strategies should be employed to promote the consumption of folate-rich foods. With current folate intakes, supplementation with folic acid is relevant especially in specific vulnerable populations, particularly in women planning and during pregnancy.

Hypothyroidism is associated with higher testosterone levels in postmenopausal women with Hashimoto's thyroiditis.

INTRODUCTION: There are few data about possible interaction of sex hormones and thyroid autoimmunity and function in women with Hashimoto's thyroiditis (HT) after menopause. Therefore, our aim was to investigate sex hormone levels in euthyroid (EuHT) and hypothyroid (HypoHT) postmenopausal women with HT. MATERIAL AND METHODS: We performed a prospective observational clinical study that included 55 women with HT (AllHT) and 18 healthy subjects (HS) after menopause matched by age, body mass index, follicle-stimulating hormone, and menopause duration. According to their thyrotropin (TSH) level, the AllHT patients were divided into two subgroups: EuHT with TSH in the range 0.35-5.5 mU/L and HypoHT with TSH above 5.5 mU/L. Total and free testosterone (T), sex hormone-binding globulin (SHBG), oestradiol (E2), and progesterone (P) were measured in all subjects. Values are presented as mean ± SD. The Mann-Whitney U test was used for comparison of values between the groups. Correlations were tested using Kendall's tau test. RESULTS: In the HypoHT group, significantly higher free T levels were found in comparison to the HS group (7.89 ± 3.55 pmol/L and 7.13 ± 3.03 pmol/L, p < 0.05). Furthermore, in HypoHT, free T was significantly higher than in EuHT (7.19 ± 5.65 pmol/L, p < 0.05). SHBG was significantly lower in HypoHT compared with HS (45.4 ± 17.4 nmol/L and 60.09 ± 19.51 nmol/L, p < 0.05). No significant correlation was found between sex hormone levels and thyroglobulin and thyroid peroxidase antibodies. CONCLUSION: We report significantly higher free and total T levels in hypothyroid postmenopausal women with HT. To our knowledge, this is the first study of sex hormone levels in postmenopausal women with HT.

Nutrihealth Study: Seasonal Variation in Vitamin D Status Among the Slovenian Adult and Elderly Population.

Several studies conducted around the world showed substantial vitamin D insufficiency and deficiency among different population groups. Sources of vitamin D in the human body include ultraviolet B (UVB)-light-induced biosynthesis and dietary intake, but people's diets are often poor in vitamin D. Furthermore, in many regions, sun exposure and the intensity of UVB irradiation during wintertime are not sufficient for vitamin D biosynthesis. In Slovenia, epidemiological data about vitamin D status in the population were investigated through a national Nutrihealth study-an extension to the national dietary survey SI.Menu (2017/18). The study was conducted on a representative sample of 125 adult (18-64 years) and 155 elderly (65-74 years old) subjects, enrolled in the study in different seasons. Their vitamin D status was determined by measuring the serum 25-hydroxy-vitamin D (25(OH)D) concentration. Thresholds for vitamin D deficiency and insufficiency were 25(OH)D levels below 30 and 50 nmol/L, respectively. Altogether, 24.9% of the adults and 23.5% of the elderly were found to be vitamin D deficient, while an insufficient status was found in 58.2% and 62.9%, respectively. A particularly concerning situation was observed during extended wintertime (November-April); vitamin D deficiency was found in 40.8% and 34.6%, and insufficient serum 25(OH)D levels were observed in 81.6% and 78.8%, respectively. The results of the study showed high seasonal variation in serum 25(OH)D levels in both the adult and elderly population, with deficiency being especially pronounced during wintertime. The prevalence of this deficiency in Slovenia is among the highest in Europe and poses a possible public health risk that needs to be addressed with appropriate recommendations and/or policy interventions.

Newborn Screening in Slovenia.

INTRODUCTION: Newborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH. METHODS: Blood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). RESULTS: From 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively. CONCLUSIONS: Successful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.