the clinical differential diagnoses of 21-hydroxylase deficiency ( 21ohd ) and cytochrome 
 p450 oxidoreductase deficiency ( pord ) are sometimes difficult since both deficiencies can 
 have similar phenotypes and high levels of 17-hydroxyprogesterone ( 17ohp ) in the blood . we 
 previously reported specific cutoff(s ) to discriminate between classic 21ohd ( c21ohd ) and 
 pord by using urinary steroid metabolites , i.e. the pregnanetriolone ( ptl)/ the cortisol 
 metabolites 5- and 5-tetrahydrocortisone ( sum of these metabolites termed thes ) ratio and 
 11-hydroxyandrosterone ( 11ohan ) , by using gas chromatography / mass spectrometry ( gc / ms ) 
 ( 1 ) . however , we did not investigate whether the 
 cutoffs were able to discriminate between non - classic 21ohd ( nc21ohd ) and pord . the 
 prevalence of nc21ohd is estimated at 1 case out of 2 million individuals in japan ( 2 ) , whereas it is reported to be 1 out of 1,000 in 
 caucasians ( 3 , 4 ) , and is considered to be the most common form of congenital adrenal hyperplasia . patients with nc21ohd have mildly impaired 21-hydroxylase activity leading to various 
 symptoms from childhood to adulthood , such as precocious pubarche , acne , hirsutism , 
 infertility , etc . biochemical diagnosis of nc21ohd is challenging because of the relatively mild 
 glucocorticoid deficiency seen in patients . we previously reported that clinically diagnosed 
 21ohd , including classic and non - classic 21ohd ( c+nc21ohd ) , can be distinguished from 
 transient hyper-17-hydroxyprogesteronemia ( th17ohp ) and controls by ptl measurements in 
 gc / ms ( 7 ) . additionally , we reported in the same study 
 that c+nc21ohd could be differentiated from pord by the ratio between 11ohan and 
 pregnanediol , which is a metabolite of progesterone , in three infants between the ages of 1 
 and 3 months ( 7 ) . the objective of this study was to investigate whether c+nc21ohd could be biochemically 
 differentiated from pord in japanese infants . in addition to ptl , thes , and 11ohan , we 
 focused on the pregnenolone ( p5 ) metabolite pregnenediol ( pd5 ) , and the aldosterone 
 metabolite tetrahydroaldosterone ( thaldo ) . we focused on these metabolites because in pord , 
 ( i ) blood p5 was shown to be higher ( 8 , 9 ) , and ( ii ) blood aldosterone and urinary thaldo were 
 shown to be normal or slightly higher , respectively , compared to that in normal subjects 
 ( 7 , 9 , 10 ) . all legal guardian(s ) gave written informed consent and the study was approved by the 
 institutional review boards at keio university hospital and keio university school of 
 medicine . we recruited 55 infants with c21ohd ( gestational age , 3541 wk ; birth weight , 
 1,6584,174 g ) , 8 infants with nc21ohd ( 3740 wk ; 2,7043,408 g ) , 16 infants with pord 
 ( 3441 wk ; 1,0183,418 g ) , 57 infants with th17ohp ( 3741 wk , 2,0624,980 g ) , and 2,473 
 controls ( 3441 wk , 7704,610 g ) . all infants were japanese with ages between 0180 d , the 
 period during which most patients with c21ohd or pord are diagnosed ( 7 , 11 ) . the diagnosis of 21ohd and 
 pord was confirmed by cyp21a2 and por gene analyses , 
 respectively . notably , all patients with nc21ohd were positive in newborn mass - screening in 
 japan . patients with 21ohd having normal genitalia and elevated dried blood spot 17ohp 
 ( positive results in newborn mass - screening ) , but without any evidence of salt wasting ( low 
 serum sodium , high serum potassium , high plasma renin activity , etc . ) the 21-deoxycortisol metabolite ptl , and the cortisol metabolites 
 5-tetrahydrocortisone and 5-tetrahydrocortisone ( hereafter referred to collectively as 
 thes ) were measured and the ratio of ptl to the cortisol metabolites ( ptl / thes ) ( 1 , 13 , 14 ) was calculated to differentiate c21ohd , nc21ohd , and 
 pord from th17ohp and control ( fig . solid arrow , steroid synthesis ; open arrow , steroid 
 metabolism ; solid line , impaired 21-hydroxylase activity ; open line , impaired 
 17-hydroxylase/17,20-lyase activity . preg , pregnenolone ; prog , 
 progesterone ; doc , deoxycorticosterone ; aldo , aldosterone ; 17ohpreg , 
 17-hydroxypregnenolone ; 11dof , 11-deoxycortisol ; dhea , dehydroepiandrosterone ; ad4 , 
 androstendione . ) . ptl was considered equal to 0.001 mg / g creatinine for calculations in infants whose 
 ptl was under the detection limit ( < 5 pg / injection ) . in addition to the metabolite of 
 11-hydroxyandrostenedione , 11ohan , ( 1 ) , we measured 
 thaldo and pd5 . these steroids and their metabolites we then calculated the 11ohan / thaldo 
 and 11ohan / pd5 ratios to discriminate between c+nc21ohd and pord ( fig . 1 ) . quantification and quality ions of each metabolite were as 
 follows ( m / z ) : ptl 449 , 359 ; 5the 488 , 578 ; 5the 488 , 
 578 ; 11ohan 448 , 358 ; thaldo 506 ( quantified ion only ) ; pd5 372 , 462 . urinary creatinine was 
 measured by iatro - lq cre ( a)ii ( lsi medience co. , tokyo , japan ) . urinary steroid 
 concentration was expressed relative to urinary creatinine ( mg / g creatinine ) . a steroid metabolic map . solid arrow , steroid synthesis ; open arrow , steroid 
 metabolism ; solid line , impaired 21-hydroxylase activity ; open line , impaired 
 17-hydroxylase/17,20-lyase activity . preg , pregnenolone ; prog , 
 progesterone ; doc , deoxycorticosterone ; aldo , aldosterone ; 17ohpreg , 
 17-hydroxypregnenolone ; 11dof , 11-deoxycortisol ; dhea , dehydroepiandrosterone ; ad4 , 
 androstendione . results of ptl and ptl / thes are shown in table 
 1table 1results of ptl and ptl / thes in c+nc21ohd , pord , th17ohp , and controls and fig . 2.urinary ptl and ptl / thes in infants with c+nc21ohd , pord , th17ohp , and controls . the upper graph is for ptl and the lower one is for ptl / thes . lines indicate 
 cutoffs : ptl 0.05 mg / g creatinine ( 010 d of age ) and 0.1 mg / g creatinine ( 11180 d 
 of age ) , ptl / thes 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) . closed circle , 
 c21ohd ; gray circle , nc21ohd ; triangle , pord ; open square , th17ohp ; dot , 
 control .. both ptl and ptl / thes showed similar overlap between c+nc21ohd , pord , th17ohp , and 
 control within 10 days of age by uniform cutoff through 0180 d of age ( ptl 0.1 and 
 ptl / thes 0.020 ) . we then separately set the cutoff for 010 d of age and 11180 d of age . ptl differentiated c+nc21ohd and pord from th17ohp and control with 100% ( 95% confidence 
 interval ( ci ) : 97.6100% ) sensitivity and 100% ( 95% ci : 99.9100% ) specificity using the 
 0.06 mg / g creatinine ( 010 d of age ) and 0.3 mg / g creatinine ( 11180 d of age ) cutoffs . ptl / thes differentiated with 100% ( 95% ci : 96.5100% ) sensitivity and 99.9% ( 95% ci : 
 99.899.9% ) specificity using the 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) 
 cutoffs . urinary ptl and ptl / thes in infants with c+nc21ohd , pord , th17ohp , and controls . the upper graph is for ptl and the lower one is for ptl / thes . lines indicate 
 cutoffs : ptl 0.05 mg / g creatinine ( 010 d of age ) and 0.1 mg / g creatinine ( 11180 d 
 of age ) , ptl / thes 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) . closed circle , 
 c21ohd ; gray circle , nc21ohd ; triangle , pord ; open square , th17ohp ; dot , 
 control . table 2table 2results of 11ohan , thaldo , and pd5 in c+nc21ohd and pord and fig . 3fig . 11ohan discriminated 
 between c21ohd and pord with 96.8% ( 95% ci : 93.396.8% ) sensitivity and 100% ( 95% ci : 
 86.1100% ) specificity using the 0.35 mg / g creatinine cutoff . although both metabolites showed 
 significantly higher distribution in pord than in c+nc21ohd ( thaldo p 
 < 0.001 , pd5 p < 0.001 ) , there was clear overlap between the two 
 groups ( table 2 ) . table 3table 3results of 11ohan / thaldo and 11ohan / pd5 in c+nc21ohd and pord and fig . 4.urinary 11ohan / thaldo and 11ohan / pd5 in infants with c21ohd , nc21ohd , and pord . the 
 upper graph is for 11ohan / thaldo and the lower one is for 11ohan / pd5 . lines indicate 
 cutoffs : 11ohan / thaldo 0.80 , 11ohan / pd5 1.0 . closed circle , c21ohd ; gray circle , 
 nc21ohd ; triangle , pord . show the results of urinary 11ohan / thaldo and 11ohan / pd5 ratio calculations . 11ohan / thaldo discriminated with 100% ( 95% ci : 97.2100% ) sensitivity and 100% ( 95% ci : 
 88.9100% ) specificity using the 0.80 cutoff . 11ohan / pd5 discriminated with 100% ( 95% ci : 
 97.2100% ) sensitivity and 100% ( 95% ci : 88.9100% ) specificity using the 1.0 cutoff . urinary 11ohan / thaldo and 11ohan / pd5 in infants with c21ohd , nc21ohd , and pord . the 
 upper graph is for 11ohan / thaldo and the lower one is for 11ohan / pd5 . lines indicate 
 cutoffs : 11ohan / thaldo 0.80 , 11ohan / pd5 1.0 . closed circle , c21ohd ; gray circle , 
 nc21ohd ; triangle , pord . results of ptl and ptl / thes are shown in table 
 1table 1results of ptl and ptl / thes in c+nc21ohd , pord , th17ohp , and controls and fig . 2.urinary ptl and ptl / thes in infants with c+nc21ohd , pord , th17ohp , and controls . the upper graph is for ptl and the lower one is for ptl / thes . lines indicate 
 cutoffs : ptl 0.05 mg / g creatinine ( 010 d of age ) and 0.1 mg / g creatinine ( 11180 d 
 of age ) , ptl / thes 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) . closed circle , 
 c21ohd ; gray circle , nc21ohd ; triangle , pord ; open square , th17ohp ; dot , 
 control .. both ptl and ptl / thes showed similar overlap between c+nc21ohd , pord , th17ohp , and 
 control within 10 days of age by uniform cutoff through 0180 d of age ( ptl 0.1 and 
 ptl / thes 0.020 ) . we then separately set the cutoff for 010 d of age and 11180 d of age . ptl differentiated c+nc21ohd and pord from th17ohp and control with 100% ( 95% confidence 
 interval ( ci ) : 97.6100% ) sensitivity and 100% ( 95% ci : 99.9100% ) specificity using the 
 0.06 mg / g creatinine ( 010 d of age ) and 0.3 mg / g creatinine ( 11180 d of age ) cutoffs . ptl / thes differentiated with 100% ( 95% ci : 96.5100% ) sensitivity and 99.9% ( 95% ci : 
 99.899.9% ) specificity using the 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) 
 cutoffs . urinary ptl and ptl / thes in infants with c+nc21ohd , pord , th17ohp , and controls . the upper graph is for ptl and the lower one is for ptl / thes . lines indicate 
 cutoffs : ptl 0.05 mg / g creatinine ( 010 d of age ) and 0.1 mg / g creatinine ( 11180 d 
 of age ) , ptl / thes 0.01 ( 010 d of age ) and 0.02 ( 11180 d of age ) . closed circle , 
 c21ohd ; gray circle , nc21ohd ; triangle , pord ; open square , th17ohp ; dot , 
 control . table 2table 2results of 11ohan , thaldo , and pd5 in c+nc21ohd and pord and fig . 3fig . 11ohan discriminated 
 between c21ohd and pord with 96.8% ( 95% ci : 93.396.8% ) sensitivity and 100% ( 95% ci : 
 86.1100% ) specificity using the 0.35 mg / g creatinine cutoff . although both metabolites showed 
 significantly higher distribution in pord than in c+nc21ohd ( thaldo p 
 < 0.001 , pd5 p < 0.001 ) , there was clear overlap between the two 
 groups ( table 2 ) . table 3table 3results of 11ohan / thaldo and 11ohan / pd5 in c+nc21ohd and pord and fig . 4.urinary 11ohan / thaldo and 11ohan / pd5 in infants with c21ohd , nc21ohd , and pord . the 
 upper graph is for 11ohan / thaldo and the lower one is for 11ohan / pd5 . show the results of urinary 11ohan / thaldo and 11ohan / pd5 ratio calculations . 11ohan / thaldo discriminated with 100% ( 95% ci : 97.2100% ) sensitivity and 100% ( 95% ci : 
 88.9100% ) specificity using the 0.80 cutoff . 11ohan / pd5 discriminated with 100% ( 95% ci : 
 97.2100% ) sensitivity and 100% ( 95% ci : 88.9100% ) specificity using the 1.0 cutoff . urinary 11ohan / thaldo and 11ohan / pd5 in infants with c21ohd , nc21ohd , and pord . the 
 upper graph is for 11ohan / thaldo and the lower one is for 11ohan / pd5 . we demonstrated that a two - step biochemical diagnosis using urinary steroid metabolites is 
 useful for diagnosis of 21ohd and pord when patients with nc21ohd were included among the 
 subjects . we propose a two - step biochemical diagnosis using ptl for the first step , and 
 11ohan / thaldo or 11ohan / pd5 for the second step , because these two markers showed no overlap 
 in each step . in the first step , we set the age - specific cutoff of ptl to differentiate c+nc21ohd and 
 pord from th17ohp and the control . we separately set the cutoff at 010 and 11180 d of age 
 because patients with 21ohd and pord who were 010 d old showed lower ptl values . assuming 
 that steroid metabolic enzyme activity in the liver was immature at early ages after birth , 
 we could explain the imperfectness of the uniform cutoff through 0180 d of age . in the second step , a downstream / upstream metabolites ratio , 11ohan / thaldo or 11ohan / pd5 , 
 was more useful than the single metabolite , 11ohan . steroid downstream / upstream metabolite 
 ratios have previously been used as markers of enzyme defects ( 15 , 16 ) . indeed , as mentioned in 
 the introduction , we had used the ratio between 11ohan and pregnanediol ( pd ) , a metabolite 
 of progesterone , for distinguishing pord from 21ohd ( 7 ) . as pd measurement is sometimes problematic in our gc / ms methods in newborns ( our 
 unpublished data ) , we used a single metabolite , 11ohan , to discriminate between c21ohd and 
 pord ( 1 ) . in this study , we chose metabolites of 
 aldosterone and pregnenolone , thaldo , and pd5 , for the following two reasons . first , 
 aldosterone and pregnenolone are upstream of 17-hydroxylase ( fig.1 ) . second , thaldo and pd5 can be measured in all newborn infants 
 ( our unpublished data ) . in this study , we recruited 0180 d old infants because most patients with c21ohd or pord 
 are diagnosed in this period ( 7 , 11 ) . although it was reported that patients with pord who were above 180 
 d of age showed similar trends in urinary steroid metabolites ( i.e. , high ptl and 
 normal - range 11ohan ) ( 11 ) , further analysis is 
 required to determine whether our cutoffs can be applicable to infants over 180 d of 
 age . this method has two advantages compared to repeated 17ohp measurement : it is a single assay 
 and offers the noninvasiveness of urine sampling . thus , this method is a potential option 
 for scrutiny of newborn mass - screened positive patients together with liquid 
 chromatography / tandem mass spectrometry ( 17 , 18 ) and genetic analysis ( 19 , 20 ) first , we do not know whether our 
 two - step method is applicable to all cases of nc21ohd . in this study , nc21ohd patients had 
 positive results in a newborn mass - screening program in japan . a few nc21ohd cases have been 
 reported to be positive in newborn mass - screening programs ( 21 ) , but most were negative because of the relatively low baseline levels of 17ohp 
 ( 5 ) . positive nc21ohd may possess less 
 21-hydroxylase activity than negatives ones ; i.e. , their ptl , 11ohan , and pd5 may be higher 
 or thes and thaldo may be lower . second , as we described in a previous study ( 1 ) , our data in japanese infants may not apply to other 
 ethnicities because of differences in common por mutations and their residual activities in 
 pord ( 9 , 22 , 
 23 ) . third , preterm infants were not included in this study whose 
 gestational age was less than 34 wk . those infants might have more immature steroid 
 metabolism in the liver , theoretically leading to lower ptl and 11ohan compared with the 
 subjects of this study . in conclusion , we demonstrated a two - step biochemical diagnosis of c+nc21ohd and pord by 
 urinary steroid profiling using ptl , thaldo , pd5 , and 11ohan .
abstract.we previously reported a two - step biochemical diagnosis to discriminate classic 
 21-hydroxylase deficiency ( c21ohd ) from p450 oxidoreductase deficiency ( pord ) by using 
 urinary steroid metabolites : the pregnanetriolone / tetrahydrocortisone ratio ( ptl / the 
 cortisol metabolites 5- and 5-tetrahydrocortisone ( sum of these metabolites termed 
 thes ) , and 11-hydroxyandrosterone ( 11ohan ) . the objective of this study was to 
 investigate whether both c21ohd and non - classic 21ohd ( c+nc21ohd ) could be biochemically 
 differentiated from pord . 
 we recruited 55 infants with c21ohd , 8 with nc21ohd , 16 with 
 pord , 57 with transient hyper-17-hydroxyprogesteronemia ( th17ohp ) , and 2,473 controls . 
 all infants were japanese with ages between 0180 d. in addition to ptl , thes , and 11ohan , 
 we measured urinary tetrahydroaldosterone ( thaldo ) and pregnenediol ( pd5 ) . the first step : 
 by ptl with the age - specific cutoffs 0.06 mg / g creatinine ( 010 d of age ) and 0.3 mg / g 
 creatinine ( 11180 d of age ) 
 , we were able to differentiate c+nc21ohd and pord from 
 th17ohp and controls ( 010 d of age : 0.06531 vs. < 0.0010.052 , 11180 d of age : 
 0.4042 vs. < 
 0.0010.086 ) with 100% sensitivity and specificity . 
 the second step : by 
 the 11ohan / thaldo or 11ohan / pd5 ratio with a cutoff of 0.80 or 1.0 , we were able to 
 discriminate between c+nc21ohd and pord ( 1.0720 vs. 0.0210.61 or 1.8160 vs. 0.0050.32 , 
 respectively ) with 100% sensitivity and specificity . 
 ptl , 11ohan / thaldo , and 11ohan / pd5 
 could differentiate between c+nc21ohd and pord in japanese infants .