Association of Blood and Urine Parameter Value Change With the Amount of Consumed Water Regime and Sample Sampling in Healthy Individuals

Background: Understanding the effect of pre-analytical factors is important for data quality of bio-specimens and health status. The study examines the effect of 9-days uid intake and 2-time sampling on concentration changes of 7-Urine and 17-Blood variables. Material and Method: SPSS software v23.0 applies to data processing. The group of 23 healthy subjects divide based on water intake and gender. Results: A statistically signicant difference(p<0.01) between 1 st /2 nd sampling is conrmed for Freezing point depression, Sodium, Potassium, Creatinine Urea and Urate in Urine and Urea, Urate, Glucose, Hematocrit, Thrombocyte in Blood. The difference between water intake after 1 st sampling is conrmed (p<0.01) for Freezing point depression, Sodium, Urate and(p<0.05) for Potassium(p<0.05), Chloride(p<0.05), Creatinine(p<0.05), Urate, Urea in Urine and Potassium(p<0.01) and Chloride(p<0.05) in Blood. Difference between gender exists for Urea(p<0.05) in Urine after 2 nd sampling and Urate(P<0.01), Glucose(p<0.01/0.05), Ht(p<0.01/0.05) after 1 st and 2 nd sampling and MCHC(p<0.01) after 2 nd sampling in Blood samples. Conclusion: Water intake increases blood and urine biomarker range after sampling. Paired T-test revealed a statistically signicant difference (P < 0.01, 95% CI), between 1st and 2nd sampling for FPD, Na + , K + , Cr, Urea, Urate in Urine and Urea, Urate, Glu, Ht, TR in Blood. Wilcoxon T-test shows the absence of statistically signicant difference (P > 0.05, α = 0.05, 95% CI) for CRP in Blood. Results are conrmed with Mann-Whitney and Kruskal-Wallis test. The difference between T/C group conrm independent T-test for FPD (P < 0.01), K + (P < 0.05), Na + (P < 0.01), Cl − (P < 0.05), Cr (P < 0.05), Urate (P < 0.01) and Urea (P < 0.05) in Urine after 1st sampling and K + (P < 0.01) and


Background
Sustainable effort must obtain for sensitivity, speci city, robustness and reproducibility of data. Preanalytical factors in uence sample quality; reproducibility, stability and false-positive results. [1,2]. Preanalytic variables include 3. categories, physiologic (age, gender, sex, time, season, altitude, menstruation, pregnancy, lifestyle like diet, caffeine, ethanol, smoking), specimens collection (overnight fasting, time of specimen collection, posture during sampling, exercise, water intake, anticoagulants-blood ratio, specimen handling and processing, added additives with anticoagulants), and in uence or interference factors (drug metabolites, laboratory tests, collection tube) affecting variable range [3].
Nervous system maintains organism homeostasis in the inward environment answering to external stimulus [4,5]. Water intake as pre-analytical variable contributes to biochemical processes and affects establishing reference range, disease diagnosis, prognosis and follow-up [6,7]. Urine and Blood samples are the most commonly used in clinical practice re ecting state of metabolome and metabolic end product [6,7]. Critical aspects of biomarker stability during clinical planning, sample collection, training, selection of sample preservation, buffers, shipping, logistics, method analysis and results in the presentation is known for the most utilized bio-specimens [8]. Studies analyzed conditions like storage time, temperature, freezing-thawing cycle on biomarker range and reproducibility [1][2][3]. Despite increasing sensitivity of methodology for determining biomarker range, discrepancies among variables exist in the literature due to sampling frequency, sample and pre-analytical factor interaction complexity [9]. Fluid intake habits and the effect on Urine/Blood biomarkers of healthy participants lack in the literature.
A current study was done as a part of the PhD thesis aim to examine the effect of 7 and 9-day regime water intake in Test (T) and Control (C) group on 7-Blood (Freezing point depression_FPD, Potassium_K + , Sodium_Na + , Chloride_Cl − , Urea, Creatinine_Cr, Urate) and 17  The regime of water intake for 23 healthy subjects included 5-days of controlled water consumption, 2days of arbitrary higher water intake in T group before 1st sampling (7th -day of water intake), 2-days of desired lover water intake in T group before 2nd sampling (9th -day of water intake), while C group drank the same level of water during 1st and 2nd sampling. Of the total M/F participants, 7-Urine and 14 Blood

Results And Discussion
Urine and Blood characteristics are present in Table 1 and Table 2. Shapiro-Wilk test indicated the presence of normality (P > 0.05) for FPD, K + , Na + , Cl − , Cr and Urate during 2nd sampling and Urea during 1st sampling in Urine samples and for Glucose, MPV during 2nd sampling, Urea, Urate, Ht, MPV during 1st sampling and K + during 1st and 2nd sampling in blood samples. All other variables showed an absence of normality (P < 0.05). All non-parametric data transform to normality using log transformation, except for CRP and additional electrolytes in the Blood. Based on this notion, the parametric and non-parametric test is employed.   Results are con rmed with Mann-Whitney and Kruskal-Wallis test. The difference between T/C group con rm independent T-test for FPD (P < 0.01), K + (P < 0.05), Na + (P < 0.01), Cl − (P < 0.05), Cr (P < 0.05), Urate (P < 0.01) and Urea (P < 0.05) in Urine after 1st sampling and K + (P < 0.01) and Cl − (P < 0.01) after 1st sampling in Blood. Figure 1. shows the difference between test and control group for 1st and 2nd sampling in Blood and Urine. Gender difference con rmed for Urea (P < 0.05) variable after 2nd sampling in Urine and for Urate (P < 0.01/0.05), Glu (P < 0.01/0.05) and Ht (P < 0.01/0.05) after 1st and 2nd sampling and MCHC (P < 0.05) during 2nd sampling in Blood.
Male sex hormones (Testosterone) affect MCHC and Ht level through the increase of erythropoietin, reduction of ferritin and hepcidin [14]. Testosterone in uence protein metabolism and the urea cycle [15].
Ex vivo cellular injury, disintegration, cellular granule release and protease activation causes alterations of cell release in vitro after sampling [1]. Hydration biomarkers in 24h urine correlate with daily total uid intake volume in sedentary adults in free-living conditions [43]. Literature ndings follow the results of the study. Explain differences in concentration changes of healthy participants, due to water intake and sample sampling.

Conclusions
Information about the water regime during 7-9 days and gender in healthy participants positively impact further clinical studies on disease patients. Water intake changes Blood and Urine biomarker concentration. Advantage of the study is clinical reproducibility, applicability, fast and precise insight into physiological changes. Results can be a reference point for protocol standardization and quality control check. Lead the improvement of healthcare service. The disadvantage is the number of participants, absence of additional information (habits, demography and genetic analysis). Future work should emphasize disadvantages by comparing health/disease states to draw clinically applicable conclusions.