Determination of 9 Amino Acid Endogenous Substances in Morning Urine of Depression Patients by LC-MS/MS / 中国药房

ABSTRACT OBJECTIVE：To establish a method for the determination of 5-hydroxyindole acetic acid（5-HIAA），glutamine， hippurate，pimelate，proline，tryptophan，tyramine，tyrosine and valine in human urine. METHODS：Morning urine samples were collected from depression patients. The sample was extracted with acetonitrile after addition of internal standard cortisone. LC-MS/ MS method was adopted. The determination was performed on XTerra RP18 column with mobile phase consisted of 0.1％ acetic acid-water as mobile phase A and 0.1％ acetic acid-acetonitrile as mobile phase B（gradient elution）. The column temperature was set at 40 ℃，and flow rate was 0.45 mL/min. Electrospray ion source（ESI）was used for quantitative analysis by multiple reaction monitoring （MRM）. The precursor/product ion transitions （m/z） were monitored at m/z 192.2→146.1，m/z 147.2→130.0，m/z 180.1→105.1，m/z 161.1→125.2，m/z 116.1→70.2，m/z 205.2→188.2，m/z 138.2→121.1，m/z 182.0→123.0，m/z 118.2→72.1 and m/z 361.2→163.0（+ion-mode）for 5-HIAA，glutamine，hippurate，pimelate，proline，tryptophan，tyramine，tyrosine，valine and cortisone，respectively. RESULTS：The linear range of 5-HIAA，glutamine，pimelate，proline，tyramine and valine were 10.00-3 200 ng/mL（r＝0.993 8-0.998 9，n＝6）. The lower limit of quantification was 10 ng/mL；the linear range of hippurate，tryptophan and tyrosine were 1 600-51 200 ng/mL（r＝0.999 2-0.999 7，n＝6）. The lower limit of quantitation was 1 600 ng/mL. The results of accuracy tests were 86.29％-98.65％（n＝6）. RSDs of intra-day and inter-day precision tests were no more than 14.65％（n＝6）CV of matrix effect were 6.18％-14.37％（n＝6）. Extraction recovery rates were 86.21％-98.14％（n＝6）. RE of stability tests were no more than 14.71％（n＝3-6）. CONCLUSIONS：The method is sensitive，accurate and suitable for the determination of 9 substances in human.

Simultaneous Determination of Ibuprofen and Indomethacin in Human Urine by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of ibuprofen and indomethacin concentration in human urine. METHODS:The urine samples were precipitated by acetonitrile. HPLC method was adopted. The determination was performed on Discovery C18 column with mobile phase consisted of acetonitrile-20 mmol/L ammonium acetate solution(85:15,V/V, pH value adjusted to 3.5 with glacial acetic acid)at the flow rate of 1.0 mL/min. UV detection wavelength was set at 220 nm. The column temperature was room temperature,and sample size was 80 μL. RESULTS:The linear range of ibuprofen and indometha-cin were both 0.1-50.0μg/mL(r=0.9996,0.9995,n=3). The limits of quantitation were both 0.1μg/mL,and the limits of detec-tion were both 0.03 μg/mL. RSDs of inter-day and intra-day were all lower than 10％(n=5),and accuracy ranged 94.7％-97.2％. The extraction recoveries of ibuprofen and indomethacin were 89.5％-91.8％ and 90.2％-92.4％(all RSDs<10％,n=15),respec-tively. CONCLUSIONS:The method is simple and rapid with high selectivity,sensitivity and accuracy. It is suitable for simultane-ous determination of ibuprofen and indomethacin concentration in human urine.

Determination of Valsartan in Human Plasma and Urine by LC-MS/MS and Its Pharmacokinetic Study / 中国药房

OBJECTIVE:To develop a method for the determination of valsartan concentration in human plasma and urine. METHODS:Plasma sample were acidified and extracted with diethyl ether for analysis,and urine sample was diluted directly for analysis. The samples were all determined by LC-MS/MS,and the separation was performed on a Aglient ZORBAX SB-C18 column with mobile phase consisted of acetonitrile and 0.1% formic acid (gradient elution) at flow rate of 0.2 ml/min. Ion transition was determined ESI ion source under multiple ion reaction monitoring with quantitative pair m/z 436.4→253.2 and qualitative ion pair m/z 436.4→291.3 for valsartan,and quantitative pair m/z 423.4→207.1 and m/z 423.4→180.2 for internal standard losartan. RE-SULTS:The linear range of valsartan were 4-5 000 ng/ml in plasma and 20-50 000 ng/ml in urine;the limit of quantification were 4 ng/ml and 20 ng/ml;plasma extraction recovery of valsartan were 61.21%-70.30%. The variation coefficient of internal standard normalized matrix effect were 3.20% and 11.21%. The within-day and between-day RSDs were no more than 8.34%. CONCLU-SIONS:The method is proved to be rapid and sensitive,and suitable for the determination of valsartan in human plasma and urine and pharmacokinetics study.

Prognostic value of procalcitonin in patients with acute paraquat intoxication / 中华急诊医学杂志

Objective To study the prognostic value of procalcitonin (PCT) level in the outcome of patients with paraquat poisoning (PQ).Methods The clinical data of 128 patients with acute PQ admitted to emergency department were collected from March 2013 through March 2014.The patients were divided into two groups:the death group and the survival group (survival of 28 days).Poisoning doses,urine concentration of PQ,time elapsed from poisoning to admission,and time elapsed from poisoning to gastrolavage were documented.And on the 1 st day,the 3rd day and the 7th day after poisoning,serum PCT were detected.The level of PCT was used to investigate the prognostic values in patients with acute PQ in the death group and survival group.Results Of 128 cases,72 (56.3％) survived and 56 died in 28 days.Among them,the level of PCT increased to some extent in the first day in 90 cases,and 48 patients died.According to trend analysis,the levels of PCT in death group on the 1st day,the 3rd day and the 7th day after PQ were significantly higher than those in survival group [ld:(0.96 ±0.13) vs.(0.08 ±0.01),3d:(1.12 ±0.14) vs.(0.28 ±0.05),7d:(1.22 ±0.14) vs.(0.20 ±0.03),P ＜0.01].There was a trend of escalating PCT levels in death group,whereas the PCT level reached the peak on the 3st day and decreased gradually in the following days in survival group.The early PCT level was obviously related to poisoning doses,urine concentration,CRP,WBC,ALT,CR (the coefficient of association were 0.794,0.723,0.724,0.332,0.700,0.414,respectively,P＜0.01).Conclusions The serum level of PCT increased in patients with acute PQ was significantly positively correlated with the oral dose and urine concentration of paraquat,and it can be used as an indicator for PQ severity.There is important clinical significance in detecting the change of serum level of PCT for estimating the condition of patients and evaluating the prognosis.

Determination of Mildronate Concentration in Human Plasma and Urine by LC-MS/MS and Pharmacokinet-ics Study / 中国药房

OBJECTIVE:To establish the method for the determination of mildronate in human plasma and urine,and to study the pharmacokinetic characteristics in healthy volunteers. METHODS:After precipitating plasma and urine sample,LC-MS/MS method was adopted. Dikma Diamonsil C18 column was used with mobile phase consisted of methanol-water(containing 0.2% for-mic acid,0.3% ammonium acetate)(31∶69,V/V)at the flow rate of 0.6 ml/min. ESI was adopted in MRM mode,by using nega-tive ion. The ion for quantitative analysis were m/z 147.10→58.20 (mildronate) and m/z 152.00→110.10 (internal standard,acet-aminophen). The pharmacokinetic parameters of mildronate with single administration and multiple administration were calculated by using DAS 2.1 software and compared. RESULTS:The linear range of mildronate in plasma were 0.02-20 ng/ml(r=0.999 3) and in urine were 0.05-40 ng/ml(r=0.998 2). The lowest limits of quantitation were 0.02 and 0.05 ng/ml. Precision and recovery met the requirements of biological specimen determination,and endogenous impurities hadn’t effect on the determination. The main pharmacokinetics parameters of low-dose,medium-dose and low-dose(250,500,750 mg)of mildronate in plasma with single ad-ministration were as follows:t1/2 were(3.39±0.81),(5.52±0.57)and(5.32±0.96)h;tmax were(0.80±0.45),(1.38±0.43)and (1.10±0.36)h;cmax were(4.17±1.46),(8.08±1.04)and(15.04±1.86)ng/ml;AUC0-36 h were(24.55±5.81),(45.50±7.07)and (85.60 ± 13.09)ng·h/ml. In the dose range,cmax,AUC0-36 h h had a linear relationship with dose (R2 were 0.974 5 and 0.968 3). The main pharmacokinetic parameters of low-dose of mildronate with multiple administration after keeping stable were as follows:cmin was(0.28 ± 0.10)ng/ml;AUCs was(38.78 ± 4.18)ng·h/ml;cs was(1.62 ± 0.17)ng/ml;DF was(3.81 ± 1.14);t1/2 was(6.17 ± 1.46)h;tmax was(1.20 ± 0.33)h;cmax was(6.46 ± 1.96)ng/ml;AUC0-36 h was(40.33 ± 4.65)ng·h/ml;accumulation factor of cmax and AUC were(1.73±0.90)and(1.64±0.40). Compared with single administration,t1/2,cmax and AUC of mildronate with multiple admin-istration after keeping stable all changed,and tmax had no signifi-cant difference. After single administration,26 h accumulative excretion rate of those groups were (0.004 009 ± 0.001 1)%, (0.004 026±0.001 01)% and(0.003 858±0.000 68)% respec-tively. CONCLUSIONS:Established method is sensitive,accurate and specific,and suitable for the determination of mildronate concentration in human plasma and urine and pharmacokinetics study. Mildronate capsule shows certain accumulation effect in healthy volunteers,and linear pharmacokinetic characteristics.

Urine Concentration and the Adaptation of Renal Medullary Cells to Hypertonicity

Hypertonicity(hypernatremia) of extracellular fluid causes water movement out of cells, while hypotonicity(hyponatremia) causes water movement into cells, resulting in cellular shrinkage or cellular swelling, respectively. In most part of the body, the osmolality of extracellular fluid is maintained within narrow range(285-295 mOsm/kgH2O) and some deviations from this range are not problematic in most tissue of the body except brain. On the other hand, the osmolality in the human renal medulla fluctuates between 50 and 1,200 mOsm/kgH2O in the process of urine dilution and concentration. The adaptation of renal medullary cells to the wide fluctuations in extracellular tonicity is crucial for the cell survival. This review will summarize the mechanisms of urine concentration and the adaptation of renal medullary cells to the hypertonicity, which is mediated by TonEBP transcription factor and its target gene products(UT- A1 urea transporter etc.).

Regulation of AQP2 in Collecting Duct: An emphasis on the Effects of Angiotensin II or Aldosterone

Vasopressin, angiotensin II (AngII), and aldosterone are essential hormones in the regulation of body fluid homeostatsis. We examined the effects of AngII or aldosterone on the regulation of body water balance. We demonstrated that 1) short-term treatment with AngII in the primary cultured inner medullary collecting duct cells played a role in the regulation of AQP2 targeting to the plasma membrane through AT1 receptor activation. This potentiated the effects of dDAVP on cAMP accumulation, AQP2 phosphorylation, and AQP2 plasma membrane targeting; 2) pharmacological blockade of the AngII AT1 receptor in rats co-treated with dDAVP and dietary NaCl-restriction (to induce high plasma endogenous AngII) resulted in an increase in urine production, a decrease in urine osmolality, and blunted the dDAVP-induced upregulation of AQP2; 3) long-term aldosterone infusion in normal rats or in rats with diabetes insipidus was associated with polyuria and decreased urine concentration, accompanied by decreased apical but increased basolateral AQP2 labeling intensity in the connecting tubule and cortical collecting duct; and 4) in contrast to the effects of dDAVP and AngII, short-term aldosterone treatment does not alter the intracellular distribution of AQP2. In conclusion, angiotensin II, and aldosterone could play a role in the regulation of renal water reabsorption by changing intracellular AQP2 targeting and/or AQP2 abundance, in addition to the vasopressin.

Regulation of Urea Transporters by Tonicity-responsive Enhancer Binding Protein

Urea accumulation in the renal inner medulla plays a key role in the maintenance of maximal urinary concentrating ability. Urea transport in the kidney is mediated by transporter proteins that include renal urea transporter (UT-A) and erythrocyte urea transporter (UT-B). UT-A1 and UT-A2 are produced from the same gene. There is an active tonicity-responsive enhancer (TonE) in the promoter of UT-A1, and the UT-A1 promoter is stimulated by hypertonicity via tonicity-responsive enhancer binding protein (TonEBP). The downregulation of UT-A2 raises the possibility that TonEBP also regulates its promoter. There is some evidence that TonEBP regulates expression of UT-A in vivo; (1) during the renal development of the urinary concentrating ability, expression of TonEBP precedes that of UT-A1; (2) in transgenic mice expressing a dominant negative form of TonEBP, expression of UT-A1 and UT-A2 is severely impaired; (3) in treatment with cyclosporine A, TonEBP was significantly downregulated after 28 days. This downregulation involves mRNA levels of UT-A2; (4) in hypokalemic animals, downregulation of TonEBP contributed to the down regulation of UT-A in the inner medulla. These data support that TonEBP directly contributes to the urinary concentration and renal urea recycling by the regulation of urea transporters.

Pathophysiology and management of disorders in water metabolism / 소아과

Even though we drink and excrete water without recognition, the amount and the composition of body fluid remain constant everyday. Maintenance of a normal osmolality is under the control of water balance which is regulated by vasopressin despite sodium concentration is the dominant determinant of plasma osmolality. The increased plasma osmolality (hypernatremia) can be normalized by the concentration of urine, which is the other way of gaining free water than drinking water, while the low plasma osmolality (hyponatremia) can be normalized by the dilution of urine which is the only regulated way of free water excretion. On the other hand, volume status depends on the control of sodium balance which is regulated mainly by renin-angiotensin-aldosterone system, through which volume depletion can be restored by enhancing sodium retention and concomitant water reabsorption. This review focuses on the urine concentration and dilution mechanism mediated by vasopressin and the associated disorders; diabetes insipidus and syndrome of inappropriate antidiuretic hormone secretion.

Long-Term Regulation of Renal Urea Transporters during Antidiuresis

To produce a concentrated urine, the renal medulla needs hypertonicity for the reabsorption of free water from collecting duct. The single effect that increases interstitial tonicity in the outer medulla is the active NaCl reabsorption in the thick ascending limb, while the single effect in the inner medulla is the passive efflux of NaCl through the thin ascending limb. The passive mechanism in the inner medulla requires high interstitial urea concentration. Two main groups of urea transporters (UT-A, UT-B) are present in the kidney, which maintains the high concentration of urea in the deepest portion of the inner medulla by intra-renal urea recycling. Recent studies suggest that UT-A1 in the terminal inner medullary collecting duct is up-regulated when urine or inner medullary interstitial urea is depleted in order to enhance the reabsorption of urea, while UT-A2 in the descending thin limb of loops of Henle and UT-B in the descending vasa recta are increased when outer medullary interstitial urea concentration is high, in order to prevent the loss of urea from the medulla to the systemic circulation, thereby increasing intra-renal urea recycling. This review will summarize the functions of the renal urea transporters in urine concentration mechanism and the recent knowledge about their long-term regulation.

Renal Transport Proteins Involved in Urinary Concentrating Mechanism / 소아과

Renal tubule and vasa recta are arranged in complex but specific anatomic relationships and the production of a concentrated urine is achieved by countercurrent multiplication mechanism in the renal medulla. This model requires that the ascending thin limb is highly permeable to NaCl but impermeable to water, while the descending thin limb is impermeable to NaCl but highly permeable to water. The single effect in the outer medulla is active NaCl reabsorption by the thick ascending limb of Henle, which is the primary energy source of urine concentration. On the other hand, the single effect for inner medullary concentration is the passive efflux of NaCl from the thin ascending limb, which requires a high concentration of interstitial urea that is reabsorbed from the terminal inner medullary collecting duct. The hypothesis has been supported by micorpuncture studies or isolated perfused tubule studies until early 1990s. In recent 10 years, many transport proteins involved in the urine concentrating mechanism have been cloned, which enabled us to understand how a concentrated urine is produced, how this process is regulated and the specific transport process that are involved, proving the countercurrent multiplication hypothesis. In this review, the transport properties of outer and inner medullary nephron segments and the transport proteins involved in the transport of NaCl, water(aquaporins), and urea(urea transporters) will be reviewed.

Determination of Ginsenoside-Rd Concentration in Human Urine by Liquid Chromatography-Mass Spectrum / 中药新药与临床药理

Objective To establish the method of liquid chromatography-mass spectrum for the determination of /Ginsenoside-Rd in human urine. Methods With the gentiopicroside as internal standard, the dilution method was adopted to treat the urine samples. Electrospmy ionization(ESI)source was applied and operated in the positive ion mode. Multiple reaction monitoring(MRM)mode was used to detect Ginsenoside-Rd content. Results By this method the linearity limit of Ginsenoside-Rd is 30.3～ 10 100 ng? mL-1, the lower quantitative limit is (30.3? 2.16) ng? mL-1, and the inter-and intra-day precision (RSD) was less than 10 % . Conclusions This is an accurate, sensitive, specific and convenient method, which can be used for the determination of Ginsenoside-Rdin urine of the healthy human.

Enuresis and Urine Concentration in Healthy Preschool Children / 대한비뇨기과학회지

PURPOSE: To determine if the urine specific gravity(SG) plays a role in enuresis, the first morning urine SG of the healthy preschool nocturnal enuretic was compared with that of the nonenuretic. The results of desmopressin were analyzed according to the pretreatment urine SG to know if the urine SG can predict the responsiveness of the medication. MATERIALS AND METHODS: Five hundred twenty healthy preschool children aged 3-6 years were entered in this prospective observer-blinded study. A comparison was made between SG of the first morning urine specimen and results of questionnaire concerning the bed wetting and voiding habits of children. The responsiveness to oral desmopressin(0.2-0.4mg h.s.) in 14-day treatment periods was also analyzed according to the urine SG in 28 children with enuresis. The responder group was defined as a reduction of at least 50% from the number of wet night. RESULTS: The incidence of enuresis was 8.6 percent. Stastistically significant difference was found between the bedwetter and nonbedwetter group with regard to the urine SG(p<0.05). The overall response rate of desmopressin was 68 percent. There was no significant difference between the responder and nonresponder group with respect to urine SG. CONCLUSIONS: The first morning urine of the enuretic showed higher probability of lower level of the SG than that of the nonenuretic. Treatment with desmopressin was associated with a significant decrease in the number of wet night, but clinical response was not predictable based on the first morning urine SG.