MINISTRY OF EDUCATION & TRAINING MINISTRY OF HEALTHHANOI MEDICAL UNIVERSITY TRAN THI NGOC ANH Urine steroid profiling by gc/ms for diagnosis of congenital steroid biosynthesis disorders
Trang 1MINISTRY OF EDUCATION & TRAINING MINISTRY OF HEALTH
HANOI MEDICAL UNIVERSITY
TRAN THI NGOC ANH
Urine steroid profiling by gc/ms for diagnosis
of congenital steroid biosynthesis disorders
in children
MEDICAL UNIVERSITY
Trang 2
Advisors : 1 PhD Tran Thi Chi Mai
2 Ass Prof PhD Tran Minh Dien
Reviewer 1: Ass Prof PhD Nguyen Thi Ha
Reviewer 2: Ass Prof PhD Nguyen Nghiem Luat
Reviewer 3: Ass Prof PhD Nguyen Phu Dat
The dissertation will be protected at the Board of University doctoral dissertation evaluation at the Hanoi Medical University:
The contents of the dissertation can be found at:
- National Library of Vietnam
- Library of Hanoi Medical University
Trang 31 Necessity of the research.
Steroid disorders in children: congenital adrenal hyperplasia is agenetic disorder with a high incidence which is early detected andtreated in newborn screening The disease is caused by acquiredmutations in a group of genes that are responsible for thebiosynthesis from three various steroid hormones includingglucocorticoids, mineralocorticoids, and sex hormones Deficiency of21-OH, 11β-OH, 3β-HSD II and 5α-reductase type 2 are theβ-HSD II and 5α-reductase type 2 are theα-reductase type 2 are thecommonest causes of such disorder and 17α-OH/17, 20-lyase,aromatase deficiency, 11β-HSD II, 17β-HSD are some rare causes.Early diagnosis and timely treatment for babies after birth areimportant to reduce mortality and complications Quantitativesteroids permits diagnosis of other forms of enzyme deficienciescausing steroid biosynthetic disorders that have been applied for over3β-HSD II and 5α-reductase type 2 are the5α-reductase type 2 are the years in the world but have not been processed in Vietnam Thenumber of patients with congenital adrenal hyperplasia and sexdevelopment disorder in Vietnam is high, especially at the NationalChildren's Hospital; it's currently managing nearly 1,000 patients, sothe quantitative analysis of steroids by gas chromatography-massspectrometry (GC-MS) might aid diagnosis and monitoringcongenital steroid biosynthetic disorders in children Before applyingnew methods to patients, it needs to be assessed, setup a referenceinterval of steroid hormones in children and apply this for diagnosisCAH and DSD
2 Purpose.
Purpose 1: Urinary steroid profiling with GC/MS requires an
appropriate validation and set out reference intervals for the urinary steroid metabolomics for children under 11 years old.
Purpose 2: Urinary steroid profiling with GC/MS to diagnose some
forms of biosynthesis of steroid hormones in the congenital adrenal cortical hyperplasia.
3 Location
The study is Department of Biochemistry - Vietnam NationalChildren's Hospital
Trang 44 New contribution of the study
GC/MS analysis of urinary steroid hormones is now the go tomethod in the first time in Vietnam and the evaluation criteria meetthe technical requirements of accuracy and authenticity Referenceintervals for 17 urinary steroids and 8 diagnostic ratios areestablished for children younger than 11 years of age Based on thismethod, diagnosis of 21-OH, 11β-OH, 3β-HSD II and 5α-reductase type 2 are theβ-HSD II and 5α-reductase type 2 are theα-reductasetype 2 deficiencies in many patients are confirmed by correspondingmutation analysis
5 Scientific and practical significance of the study.
The research is highly practical and contributes to providing amodern diagnostic method to detect steroid biosynthetic disorders.This method permits diagnosis of other forms of enzyme deficienciescausing congenital adrenal hyperplasia, thereby reducing the number
of cases that require genetic analysis to reduce the cost of diagnosisand treatment
This research has scientific significance with tightly-packed layout,appropriate data processing methods, international standards for newtechnical implementation with the method of evaluation, reference aninterval which is applied to the diagnosis of congenital adrenalhyperplasia and sex development disorder
Theme of study is innovative, trendy and up-to-date whichsuccessfully implements quantitative urinary steroids method in thefirst time in Vietnam, and it has been routinely applied to patientssince 2018
4 The structure of thesis.
- The thesis is presented in 13β-HSD II and 5α-reductase type 2 are the6 pages (excluding references and
appendices) It is divided into 7 parts: 2 pages of Introduction;
Chapter 1: 40 pages of Overview; chapter 2: 16 pages of Subjects andresearch methods; Chapter 3β-HSD II and 5α-reductase type 2 are the: 46 pages of Results; chapter 4: 29pages of Discussion; 2 pages of Conclusion; 1 page ofRecommendation
- The thesis consists of 3β-HSD II and 5α-reductase type 2 are the4 tables, 3β-HSD II and 5α-reductase type 2 are the4 charts, figures and diagrams,
129 references, including 20 documents in Vietnamese and 109documents in English The appendix includes: Sample survey andevaluation forms; collective form, results of genetic analysis; list ofpatients participating in the study
Trang 5Chapter 1: OVERVIEW
1 Pathology of steroid biosynthetic disorders
Deficiency of any enzyme responsible for the biosynthesis alsodisrupts steroid biosynthesis, which is divided into three maingroups, including congenital adrenal hyperplasia (CAH), disorders ofelectrolyte, and disorders of sex development (DSD) The mostcommon cause is deficiency of 21-hydroxylase enzyme accounted formore than 90% of cases of CAH, deficiency of 11β-OH rankedsecond with 5α-reductase type 2 are the-8%, deficiency of 3β-HSD II and 5α-reductase type 2 are theβ-HSD II and other enzymes arerare causes of CAH In addition, deficiency of enzyme 5α-reductase type 2 are theα-reductasetype 2 causes a reduction in the synthesis of 5α-reductase type 2 are theα-dihydrotestosterone,resulting the feminisation of men-children, which is one of the twomost common causes of DSD in men-children with 46,XYchromosomes
Quantification of 17-OH-P from a dried blood spot helps to detectcases of typical CAH due to deficiency of 21-OH, and some patientswith deficiency of 11β-OH, however, it cannot help to detect cases ofCAH and DSD due to deficiency of 3β-HSD II and 5α-reductase type 2 are theβ- HSD II, Cyt P45α-reductase type 2 are the0oxidoreductase (POR), 5α-reductase type 2 are theα-reductase type 2, AME (apparentmineralocorticoid excess) At the same time, 17-OHP test shows ahigh false-positive in preterms, low birth weight newborn and it isirrespective of CAH types also
2 GC/MS analysis of urinary steroid quantification
Urinary steroid quantification by Selective Ion Monitoring GasChromatography-Mass Spectrometry (GC/MS-SIM) has been appliedover the world since the 1980s to date in diagnosing forms ofcongenital adrenal hyperplasia The method has high sensitivity andspecificity and urine specimens, so it is suitable for all subjects,especially children Each pathology of enzyme deficiency has acharacteristic chromatographic pattern due to a deficiency ofhormones and an increase of precursor in previous position ofenzyme The concentration of steroids compared with the referenceintervals and the diagnostic precursor-to-product ratios are used todetect and distinguish different disorders of biosynthesis of steroidhormones due to deficiency of 21-OH, 11β-OH, 3β-HSD II and 5α-reductase type 2 are theβ-HSD II or 5α-reductase type 2 are theα-reductase type 2 and deficiency of 17-OH, POR, AME
Trang 6Urinary steroid quantification was first implemented in Vietnam,
so it is necessary to evaluate the method and set out the referenceintervals according to international guidelines to ensure the quality ofmethod and comply with ISO 15α-reductase type 2 are the189
Technical process is improved under the guidance of Honour JW,Ronda F Greaves In patients with clinical signs of CAH and DSD,
we quantify the concentration of steroid hormones met the diagnosticcriteria of steroid biosynthetic disorders and then collect the results ofelectrolytes and ACTH in the medical record and a genetic analysis issuggested to confirm the diagnosis
Chapter 2 SUBJECTS AND METHODS OF RESEARCH
2.1 Subjects of Research
2.1.1 Control group:
Including 269 healthy children aged from 1 day to 11 years,divided into 4 groups:
Group 1: 64 children from 1 day to 28 days old
Group 2: 61 children from 1 month (≥ 29 days) to < 2 yearsold
Group 3β-HSD II and 5α-reductase type 2 are the: 74 children from 2 years old to < 8 years old
Group 4: 70 children aged 8-< 11 years
Sex: Masculine and feminine ratio is equal
Selection criteria: Healthy children do not have abnormal signs ofCAH and sex development disorder Full-term infant according toWHO standards within each gestational term of 3β-HSD II and 5α-reductase type 2 are the7-42 weeks, birthweight > 25α-reductase type 2 are the00g, with no family history of a disorder Specimenswere collected at the Department of Obstetrics and Gynecology ofVinh Phuc General Hospital, the commune or ward health clinicswhere carríe out the routine vaccination program for children,kindergartens and primary schools in Vinh Phuc province and HanoiCity and from groups of disease in the meanwhile
Exclusion criteria: The family and/or the children do not agree toparticipate in the study, the child has one of the clinical signssuspecting the above mentioned CAH or DSD or receivingcorticosteroids within 1 month
Trang 72.1.2 Group of diseases:
Including 200 patients suspected of having a steroid biosyntheticdisorders, diagnosed with CAH, DSD, and ED by endocrinologistsand pediatricians
Location: Department of Endocrinology - Metabolism - Genetics
of Vietnam National Children's Hospital and Department of PediatricSurgery - Viet Duc Hospital
Sampling time: from October 2015α-reductase type 2 are the to July 2018
Selection criteria: when there are one or more signs:
Patients with CAH: Clinical and laboratory standards based on
announced New MI standards
Clinical symptoms: Ambiguous genitalia, genitals may not appearclearly male or female children; signs of early puberty in boys; signs
of dehydration, adrenal insufficiency in both men and women
Patients with disorders of sex development:
There are symptoms: Genital abnormalities such as micropenis,narrowing of the urethra, hidden testicles, penile agenesis, doublescrotum and female external organs Bodies of male children toappear feminized such as breast development, menstruation, signs ofprimary amenorrhea in female appearance
Families and/or patients suspected of having CAH and DSDconsented and signed an informed consent form to participate into theresearch group
Exclusion criteria: patients and/or families do not agree to
participate in the study
Patients with electrolyte disorders merely due to systemic diseases,digestive organs without clinical signs CAH, DSD
Patients with dysplasia steroid biosynthetic disorders such as related Cushing syndrome, Addison, adrenal adenoma
drug-Specimens: Random urine specimens were stored for 2 weeks at
2-80C, 6 months at -200C
2.3.3 Method of Research
- Validation of GC-MS urinary steroid method based on
Trang 8Westgard's instructions with the following contents: Determine thelimits of detection, recovery, imprescision and and acuracy.
- Establish the urinary steroid reference intervals in childrenaccording to the guidelines of the International Federation of ClinicalChemistry (IFCC) and Clinical and Laboratory Standards Institute(CLSI): Evaluate the distribution of each urinary steroid, calculatethe reference intervals for men and women in each age group which
be set for both men and women in case of without difference.Calculate the diagnostic ratio, set out the concentration intervals anddiagnostic ratios for men and women in each age group If there is nodifference the concentration intervals will be common for both menand women Reference interval calculated by non-parametric methodwith non-standard distribution data Arrange the data in theincreasing direction of the value, select the low value and the highvalue of the reference interval from the list of referenced values sothat the low reference value corresponds to the 2.5α-reductase type 2 are the percentile (2.5α-reductase type 2 are the%percentile) and high reference value corresponding to percentile 97.5α-reductase type 2 are the(97.5α-reductase type 2 are the% percentile) of the reference interval
x0,025α-reductase type 2 are the = 0.025α-reductase type 2 are the (n + 1)
x0,975α-reductase type 2 are the = 0.975α-reductase type 2 are the (n + 1)
In which:
x 0.025α-reductase type 2 are the is the low reference value corresponding to 2.5α-reductase type 2 are the percentile
x0.975α-reductase type 2 are the is the high reference value corresponding to the 97.5α-reductase type 2 are thepercentile
n is the number of samples that set out for reference interval
- Develop standards for diagnosis and application of urinarysteroid quantification in the diagnosis and monitoring of steroidbiosynthetic disorders in patients suspected of having CAH or DSD.-Collect results of blood tests, genetic analysis in medical records.Diagnostic criteria were established for a number of conditions of steroid enzyme deficiency based on Krone et al., Chan OK et al in the laboratory's existing condition
Diagnosis of 21-OH deficiency when there are the following
Trang 9 There is the appearance of peak 17OHPN, PTL onchromatogram (qualitative)
The PT concentration is higher than the reference intervals
The ratio of PT/ (THE +THF +5α-reductase type 2 are theα-THF) is higher than thereference intervals
The ratio of THS/ (THE+THF+5α-reductase type 2 are theα-THF) in the referenceintervals
Diagnosis of 11β-OH deficiency when there are the following
characteristics:
There is a clear appearance of THS peak on chromatogram(qualitative)
The concentration of THS is higher than the reference intervals
The ratio THS/ (THE +THF +5α-reductase type 2 are the α -THF) is higher than thereference
Diagnosis of 3β-HSD II deficiency when there are the following
characteristics:
DHEA levels are higher than the reference intervals
The ratio of DHEA/ (THE +THF +5α-reductase type 2 are the α-THF) is higher than thereference intervals
There are no signs of deficiency of 21-OH or 11β-OH whenurinary steroid quantification is conducted
Diagnosis of 5α-reductase type 2 deficiency when there are the
2.3.7 Analyzing and processing data.
Use Excell, SPSS 22.0 software, Method validation software in data
processing and method comparison
Trang 11Chapter 3: RESEARCH RESULTS 3.1 Results of method validation
Table 3.1: Limit of quantitative and recovery
Numbe
Recovery (μmol/L)%)
1 An 0.11 13β-HSD II and 5α-reductase type 2 are the.18 102,5α-reductase type 2 are the
2 Et 0.05α-reductase type 2 are the 10.69 114,73β-HSD II and 5α-reductase type 2 are the DHEA 0.10 17.84 113β-HSD II and 5α-reductase type 2 are the,3β-HSD II and 5α-reductase type 2 are the
4 11 keto An 0.12 8.3β-HSD II and 5α-reductase type 2 are the6 111,15α-reductase type 2 are the 11-OH An 0.07 19.3β-HSD II and 5α-reductase type 2 are the 112,0
6 11-OH Et 0.09 10.48 113β-HSD II and 5α-reductase type 2 are the,6
8 PT 0.07 16.86 101,5α-reductase type 2 are the
9 A’3β-HSD II and 5α-reductase type 2 are the 0.21 9.82 91,4
11 THF 0.11 18.43β-HSD II and 5α-reductase type 2 are the 110,2
12 5α-reductase type 2 are theα-THF 0.11 18.07 106,3β-HSD II and 5α-reductase type 2 are the13β-HSD II and 5α-reductase type 2 are the α-Cortolone 0.09 8.3β-HSD II and 5α-reductase type 2 are the2 91,3β-HSD II and 5α-reductase type 2 are the
14 β-Cortol 0.11 15α-reductase type 2 are the.16 91,815α-reductase type 2 are the β-Cortolone 0.06 10,9 99,4
16 α-Cortol 0.27 13β-HSD II and 5α-reductase type 2 are the,09 90,9Quantitative limits of 14/16 steroids ranged from 0.05α-reductase type 2 are the to 0.16Smol/L, particularly α-Cortol and A'3β-HSD II and 5α-reductase type 2 are the had quantitative limits of 0.27and 0.21 (Smol/L) respectively which is higher than other productsbut less valuable in diagnosing congenital adrenal hyperplasia Recovery of steroid metabolic products is from 90% to 115α-reductase type 2 are the%.The average is 101.9 ± 8.4% ( X ± SD).
Trang 12Table 3.2 Short term impresision
X
(µmol/L)
SD(µmol/L)
CV(%)
X
(µmol/L)
SD(µmol/L)
CV(%)
An 1.25α-reductase type 2 are the 0.096 7.72 18.44 1.695α-reductase type 2 are the 9.20
Et 1.5α-reductase type 2 are the0 0.098 6.5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are the 12.63β-HSD II and 5α-reductase type 2 are the 1.091 8.64
PD 2.3β-HSD II and 5α-reductase type 2 are the1 0.06 2.72 2.61 0.093β-HSD II and 5α-reductase type 2 are the 3β-HSD II and 5α-reductase type 2 are the.5α-reductase type 2 are the4
PT 1.49 0.015α-reductase type 2 are the 1.01 86.87 0.677 0.78THE 7.47 0.3β-HSD II and 5α-reductase type 2 are the14 4.20 22.3β-HSD II and 5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are the 1.3β-HSD II and 5α-reductase type 2 are the62 6.10THF 2.87 0.107 3β-HSD II and 5α-reductase type 2 are the.72 6.15α-reductase type 2 are the 0.3β-HSD II and 5α-reductase type 2 are the5α-reductase type 2 are the5α-reductase type 2 are the 5α-reductase type 2 are the.805α-reductase type 2 are theα-THF 3β-HSD II and 5α-reductase type 2 are the.03β-HSD II and 5α-reductase type 2 are the 0.13β-HSD II and 5α-reductase type 2 are the2 4.3β-HSD II and 5α-reductase type 2 are the4 8.02 0.45α-reductase type 2 are the9 5α-reductase type 2 are the.70α-Cortolone 3β-HSD II and 5α-reductase type 2 are the.13β-HSD II and 5α-reductase type 2 are the 0.05α-reductase type 2 are the1 1.62 4.42 0.090 2.04β-Cortol 1.22 0.05α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are the 2.82 1.23β-HSD II and 5α-reductase type 2 are the 0.041 3β-HSD II and 5α-reductase type 2 are the.3β-HSD II and 5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are theβ-Cortolone 2.13β-HSD II and 5α-reductase type 2 are the 0.045α-reductase type 2 are the 2.11 3β-HSD II and 5α-reductase type 2 are the.16 0.075α-reductase type 2 are the 2.3β-HSD II and 5α-reductase type 2 are the6α-Cortol 0.49 0.03β-HSD II and 5α-reductase type 2 are the0 6.12 0.80 0.03β-HSD II and 5α-reductase type 2 are the1 3β-HSD II and 5α-reductase type 2 are the.95α-reductase type 2 are the
Short-term accuracy of urinary steroid metabolic productswith normal specimens has CV from 1.01 to 7.72%
Short-term accuracy of urinary steroid metabolic productswith pathological specimens has a CV of 0.78 to 9.72%
The median value of CV in normal specimens is 4.0 ± 2.2(%) and pathological specimens is 4.7 ± 2.7 (%) ( X ± SD).
Table 3.3 Long term impresision
Trang 13X
(µmol/L)
SD(µmol/L)
CV(%)
An 1.23β-HSD II and 5α-reductase type 2 are the 0.15α-reductase type 2 are the 12.5α-reductase type 2 are the5α-reductase type 2 are the 15α-reductase type 2 are the.00 2.44 16.26
Et 1.48 0.18 11.87 10.14 1.61 15α-reductase type 2 are the.91
PD 2.42 0.3β-HSD II and 5α-reductase type 2 are the4 14.04 2.5α-reductase type 2 are the0 0.3β-HSD II and 5α-reductase type 2 are the5α-reductase type 2 are the 14.13β-HSD II and 5α-reductase type 2 are the
PT 1.72 0.23β-HSD II and 5α-reductase type 2 are the 13β-HSD II and 5α-reductase type 2 are the.61 89.3β-HSD II and 5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are the 11.17 12.5α-reductase type 2 are the1THE 8.08 1.04 12.9 22.96 3β-HSD II and 5α-reductase type 2 are the.68 16.01THF 3β-HSD II and 5α-reductase type 2 are the.14 0.3β-HSD II and 5α-reductase type 2 are the2 10.16 6.3β-HSD II and 5α-reductase type 2 are the4 0.87 13β-HSD II and 5α-reductase type 2 are the.75α-reductase type 2 are theα-THF 3β-HSD II and 5α-reductase type 2 are the.46 0.48 13β-HSD II and 5α-reductase type 2 are the.81 8.48 1.23β-HSD II and 5α-reductase type 2 are the 14.5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are theα-Cortolone 3β-HSD II and 5α-reductase type 2 are the.3β-HSD II and 5α-reductase type 2 are the6 0.40 11.83β-HSD II and 5α-reductase type 2 are the 5α-reductase type 2 are the.19 0.78 15α-reductase type 2 are the.7β-Cortol 1.3β-HSD II and 5α-reductase type 2 are the9 0.20 14.5α-reductase type 2 are the3β-HSD II and 5α-reductase type 2 are the 1.47 0.22 15α-reductase type 2 are the.26β-Cortolone 2.3β-HSD II and 5α-reductase type 2 are the7 0.3β-HSD II and 5α-reductase type 2 are the0 12.65α-reductase type 2 are the 3β-HSD II and 5α-reductase type 2 are the.5α-reductase type 2 are the6 0.47 13β-HSD II and 5α-reductase type 2 are the.14α-Cortol 0.5α-reductase type 2 are the8 0.08 13β-HSD II and 5α-reductase type 2 are the.3β-HSD II and 5α-reductase type 2 are the7 0.85α-reductase type 2 are the 0.14 16.95α-reductase type 2 are the
The median value ± SD of CV in normal specimens is 12.85α-reductase type 2 are the ± 1.24
(%) and pathological specimens is 14.86 ± 1.4 (%) ( X ± SD).
Trang 14Table 3.4: Correlation bethwen results and median of EQA
An 0.969 -0.015α-reductase type 2 are the y=0.969x-0.015α-reductase type 2 are the 0.991
Et 1.014 -0.024 y=1.014x-0.024 0.992DHEA 1.25α-reductase type 2 are the0 0.0 y=1.25α-reductase type 2 are thex + 0 0.983β-HSD II and 5α-reductase type 2 are the11Keto An 1.106 -0.065α-reductase type 2 are the y=1.106x-0.065α-reductase type 2 are the 0.99111-OH An 1.042 -0.068 y=1.0x-0.068 0.996
11-OH Et 0.611 0.03β-HSD II and 5α-reductase type 2 are the9 y=0.611x+0.03β-HSD II and 5α-reductase type 2 are the
Cortolone 1.124 -0.211 y=1.124x-0.211 0.981β-
Cortolone 1.113β-HSD II and 5α-reductase type 2 are the - 0.082 y=1.113β-HSD II and 5α-reductase type 2 are thex-0.082 0.992α-Cortol 1.00 -0.01 y=1.0x-0.01 0.997β-Cortol 1.013β-HSD II and 5α-reductase type 2 are the -0.006 y=1.013β-HSD II and 5α-reductase type 2 are thex-0.006 0.994The urinary steroids are closely correlated with the medianvalues of the EQA results with a correlation coefficient r of 0.97-0.999 The angle coefficient approximates the value 1.0 andintersects the vertical axis at point b with a value of approximately 0,
so the linear lines almost go through the origin THE and DHEA arehigher than EQA value with a coefficient of 1.25α-reductase type 2 are the and 11-OH Et is
Trang 15lower than external quality assessment value with a coefficient of0.611 The urinary steroids have agreement similarity to the medianvalue of external quality assessment.
3.2 Reference intervals of steroids in chidren
Table 3.5: Age and sex in controls
8-11 years 29 41.4 41 5α-reductase type 2 are the8.9 0.15α-reductase type 2 are the1
Sum 13β-HSD II and 5α-reductase type 2 are the7 5α-reductase type 2 are the0.9 13β-HSD II and 5α-reductase type 2 are the2 49.1 0.243β-HSD II and 5α-reductase type 2 are theThere is no difference in the number of boys and girls in each agegroup as well as with the total number of men and women (p> 0.05α-reductase type 2 are the)
Distribution of urinary steroid metabolic products
All 16 parameters of steroid levels do not follow the Gaussiandistribution Comparison is based on median value (med.), lowestvalue (min), highest value (max) The reference intervals comprising95α-reductase type 2 are the% of the median value is taken at the cutoff points of 2.5α-reductase type 2 are theth and97.5α-reductase type 2 are theth percentiles
The concentration of An, Et, 11OH An and 11OH Et is verylow in children < 8 years of age, increasing gradually in childrenaged 8-11 years Concentration of THE, THF, 5α-reductase type 2 are theα-THF increases withage