disorder of mineral and bone metabolism is one of the most prevalent and serious abnormalities in dialysis patients .
second - generation parathyroid hormone ( pth ) assays , also called intact pth assays , have been widely used for optimal management of parathyroid function and bone remodelling [ 13 ] . until recently ,
these assays were believed to react only with full - length pth ( 184 ) ; however , they also detect large c - terminal fragments with a partially preserved n - terminal structure , mainly pth ( 784 ) .
this is especially relevant in dialysis patients because reduced clearance from the kidneys may result in increased levels of these fragments .
moreover , pth ( 784 ) has been shown to antagonize the calcaemic and bone - resorbing effects of pth ( 184 ) [ 7 , 8 ] .
third - generation pth assays , such as the whole pth assay or bio - intact pth assay , have recently been developed to overcome such complex methodological and biological problems .
pth values obtained from intact pth assays are generally higher than those obtained from the whole pth assay because the latter does not detect non-(184 ) fragments [ 1013 ] .
rare exceptions to this rule have been reported in patients with severe primary and secondary hyperparathyroidism and parathyroid cancer [ 1419 ] . moreover
, a new molecular form of n - pth , distinct from pth ( 184 ) , has been identified in the circulation after hplc fractionation of serum [ 1416,18 ] .
we present a series of cases with progressive hyperparathyroidism in whom whole pth levels were paradoxically higher than intact pth levels .
we then reviewed the recent literature and summarized the clinical features of published cases with the reversed whole pth / intact pth ratio , which suggested that this reversed ratio could be a marker for the severity of hyperparathyroidism .
a 55-year - old male had been receiving haemodialysis since february 2003 for end - stage renal disease ( esrd ) due to diabetic nephropathy . in may 2006 , on detection of high intact pth levels ( 327 pg / ml ; elecsys pth ; roche diagnostics , mannheim , germany )
the patient was treated with intravenous calcitriol ( 1.0 g / week ) . however , serum intact pth levels progressively increased to 528 pg / ml and abnormally elevated levels of whole pth ( 614 pg / ml ; whole pth ; scantibodies laboratories , santee , ca , usa ) were found .
doppler ultrasonography showed a single enlarged hypervascular parathyroid gland ( 14 mm  11 mm  7 mm ) .
surgical parathyroidectomy was performed , and the intact and whole pth levels decreased to 17 and 5.9 pg / ml , respectively , with normalization of the reversed whole pth / intact pth ratio ( figure 1 ) .
serum pth levels before and after parathyroidectomy . reversed whole pth / intact pth ratio normalized after surgery , as is usual in uraemic patients .
the resected gland showed hyperplasia of the parathyroid cells without the normal rim , supporting the diagnosis of secondary hyperparathyroidism ( figure 2a ) .
however , the complication of sporadic primary adenoma remains a possibility in dialysis patients , especially those with a single enlarged gland .
thus , we performed immunohistochemical analysis , which is known to be helpful when differentiating these two disorders , as previously reported by tominaga et al . .
the immunostaining pattern of ki-67 indicated accelerated cell growth progression ( labelling index 28/1000 ) ( figure 2b ) , while heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 was not consistent with primary adenoma ( labelling index 360/1000 ) ( figure 2c ) .
thus , the gland was diagnosed as a single nodule , the most advanced type of parathyroid hyperplasia in chronic uraemic patients .
( b ) immunohistochemical findings of ki-67 indicate accelerated cell growth progression ( labelling index 28/1000 ) .
( c ) heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 ( labelling index 360/1000 ) suggesting a single nodule of uraemic hyperparathyroidism rather than a sporadic primary adenoma .
a 58-year - old female had been receiving haemodialysis since may 1996 for esrd due to iga nephropathy . in february 2007 , despite maintenance of intact pth levels ( 178 pg / ml ; elecsys pth ) and whole pth levels ( 134 pg / ml ) , routine doppler ultrasonography revealed a hypervascular enlarged gland ( 14 mm  12 mm  12 mm ) . with the intention of preventing the progression of secondary hyperparathyroidism , oral falecalcitriol ( 0.15 g / day ) was started ; however , after 10 months of treatment , serum intact and whole pth levels progressively increased to 685 and 704 pg / ml , respectively , with the reversed whole pth / intact pth ratio .
re - examination by ultrasonography showed a progressive enlargement of the parathyroid gland ( 18 mm  14 mm  14 mm ) ( figure 3a ) , and a hot spot was detected at the same location by tc methoxyisobutyl - isonitrile scintigraphy ( figure 3b ) .
serum bone alkaline phosphatase levels increased up to 102 u / l , indicating extremely high bone turnover .
accordingly , the patient was scheduled for parathyroidectomy . ( a ) power doppler ultrasonography showing a single enlarged hypervascular parathyroid gland ( 18 mm  14 mm  14 mm ) .
( b ) tc methoxyisobutyl - isonitrile scintigraphy demonstrating a hot spot in the right inferior parathyroid gland .
a 60-year - old male had been receiving haemodialysis since november 2001 for esrd due to diabetic nephropathy . for several years
, serum intact pth levels were maintained between 200 and 300 pg / ml ( elecsys pth ) .
however , in june 2006 , despite ideal control of parathyroid function , a routine neck ultrasonography examination revealed an enlarged hypervascular parathyroid gland ( 17 mm  14 mm  15 mm ) .
subsequently , secondary hyperparathyroidism rapidly advanced , and in december 2007 , serum intact and whole pth levels increased to 612 and 801 pg / ml , respectively , with the reversed whole pth / intact pth ratio .
significantly progressive enlargement of the parathyroid gland ( 23 mm  21 mm  21 mm ) was shown by ultrasonography .
a 54-year - old male had been receiving haemodialysis since 1974 for esrd due to chronic glomerulonephritis . since 1981
, he was found to have extremely high carboxyl - terminal pth ( c - pth ) levels between 20 and 30 ng / ml ( normal range , 0.21.0 ng / ml ) .
total parathyroidectomy with forearm autograft was performed in 1986 , and the c - pth levels effectively decreased to 0.5 ng / ml . the resected gland showed parathyroid hyperplasia .
for about two decades after surgery , secondary hyperparathyroidism , which was evaluated by intact pth assays , had been managed well ; however , in november 2007 , abnormally higher whole pth levels ( 358 pg / ml ) than intact pth levels ( 278 pg / ml ; total pth ; scantibodies laboratories ) were found .
ultrasonography of the forearm showed an enlarged parathyroid tissue ( 17 mm  7 mm  3 mm ) .
after informed consent was obtained , we performed a simplified casanova test ( total ischaemic blockade of the graft - bearing arm ) , as previously described .
following temporary ischaemic autograftectomy , the intact and whole pth levels significantly decreased to 127 and 117 pg / ml , respectively , with normalization of the reversed whole pth / intact pth ratio , suggesting that the autografted parathyroid tissue was the cause of the reversed ratio .
a 39-year - old female had been receiving haemodialysis since 1979 for esrd due to chronic glomerulonephritis . in 1993 ,
total parathyroidectomy with forearm autograft was performed for secondary hyperparathyroidism , which was then maintained for several years .
however , in january 2007 , elevated levels of intact pth ( 396 pg / ml ; elecsys pth ) , whole pth ( 263 pg / ml ) and bone alkaline phosphatase activity ( 63 u / l ) were found .
ultrasonography of the graft - bearing arm showed an enlargement of the parathyroid tissue ( 17 mm  16 mm  5 mm ) . when pth levels were measured in the sera obtained from the graft - bearing arm , extremely elevated levels of intact and whole pth levels
were found ( 3600 and 4490 pg / ml , respectively ) , with the reversed whole pth / intact pth ratio .
, the intact and whole pth levels measured in the sera obtained from the graft - bearing arm decreased to 27 and 39 pg / ml , respectively , but without normalization of the reversed ratio .
it was then suggested that small fragments of the parathyroid tissue ( i.e. the probable cause of the reversed whole pth / intact pth ratio ) remained in the forearm .
a 55-year - old male had been receiving haemodialysis since february 2003 for end - stage renal disease ( esrd ) due to diabetic nephropathy . in may 2006 , on detection of high intact pth levels ( 327 pg / ml ; elecsys pth ; roche diagnostics , mannheim , germany )
the patient was treated with intravenous calcitriol ( 1.0 g / week ) . however , serum intact pth levels progressively increased to 528 pg / ml and abnormally elevated levels of whole pth ( 614 pg / ml ; whole pth ; scantibodies laboratories , santee , ca , usa ) were found .
doppler ultrasonography showed a single enlarged hypervascular parathyroid gland ( 14 mm  11 mm  7 mm ) .
surgical parathyroidectomy was performed , and the intact and whole pth levels decreased to 17 and 5.9 pg / ml , respectively , with normalization of the reversed whole pth / intact pth ratio ( figure 1 ) .
serum pth levels before and after parathyroidectomy . reversed whole pth / intact pth ratio normalized after surgery , as is usual in uraemic patients .
the resected gland showed hyperplasia of the parathyroid cells without the normal rim , supporting the diagnosis of secondary hyperparathyroidism ( figure 2a ) .
however , the complication of sporadic primary adenoma remains a possibility in dialysis patients , especially those with a single enlarged gland .
thus , we performed immunohistochemical analysis , which is known to be helpful when differentiating these two disorders , as previously reported by tominaga et al . .
the immunostaining pattern of ki-67 indicated accelerated cell growth progression ( labelling index 28/1000 ) ( figure 2b ) , while heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 was not consistent with primary adenoma ( labelling index 360/1000 ) ( figure 2c ) .
thus , the gland was diagnosed as a single nodule , the most advanced type of parathyroid hyperplasia in chronic uraemic patients .
( b ) immunohistochemical findings of ki-67 indicate accelerated cell growth progression ( labelling index 28/1000 ) .
( c ) heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 ( labelling index 360/1000 ) suggesting a single nodule of uraemic hyperparathyroidism rather than a sporadic primary adenoma .
a 58-year - old female had been receiving haemodialysis since may 1996 for esrd due to iga nephropathy . in february 2007
, despite maintenance of intact pth levels ( 178 pg / ml ; elecsys pth ) and whole pth levels ( 134 pg / ml ) , routine doppler ultrasonography revealed a hypervascular enlarged gland ( 14 mm  12 mm  12 mm ) . with the intention of preventing the progression of secondary hyperparathyroidism , oral falecalcitriol ( 0.15 g / day )
was started ; however , after 10 months of treatment , serum intact and whole pth levels progressively increased to 685 and 704 pg / ml , respectively , with the reversed whole pth / intact pth ratio .
re - examination by ultrasonography showed a progressive enlargement of the parathyroid gland ( 18 mm  14 mm  14 mm ) ( figure 3a ) , and a hot spot was detected at the same location by tc methoxyisobutyl - isonitrile scintigraphy ( figure 3b ) .
serum bone alkaline phosphatase levels increased up to 102 u / l , indicating extremely high bone turnover .
( a ) power doppler ultrasonography showing a single enlarged hypervascular parathyroid gland ( 18 mm  14 mm  14 mm ) .
( b ) tc methoxyisobutyl - isonitrile scintigraphy demonstrating a hot spot in the right inferior parathyroid gland .
a 60-year - old male had been receiving haemodialysis since november 2001 for esrd due to diabetic nephropathy . for several years , serum intact pth levels were maintained between 200 and 300 pg / ml ( elecsys pth ) .
however , in june 2006 , despite ideal control of parathyroid function , a routine neck ultrasonography examination revealed an enlarged hypervascular parathyroid gland ( 17 mm  14 mm  15 mm ) .
subsequently , secondary hyperparathyroidism rapidly advanced , and in december 2007 , serum intact and whole pth levels increased to 612 and 801 pg / ml , respectively , with the reversed whole pth / intact pth ratio .
significantly progressive enlargement of the parathyroid gland ( 23 mm  21 mm  21 mm ) was shown by ultrasonography .
a 54-year - old male had been receiving haemodialysis since 1974 for esrd due to chronic glomerulonephritis . since 1981 , he was found to have extremely high carboxyl - terminal pth ( c - pth ) levels between 20 and 30 ng / ml ( normal range , 0.21.0 ng / ml ) .
total parathyroidectomy with forearm autograft was performed in 1986 , and the c - pth levels effectively decreased to 0.5 ng / ml . the resected gland showed parathyroid hyperplasia .
for about two decades after surgery , secondary hyperparathyroidism , which was evaluated by intact pth assays , had been managed well ; however , in november 2007 , abnormally higher whole pth levels ( 358 pg / ml ) than intact pth levels ( 278 pg / ml ; total pth ; scantibodies laboratories ) were found .
ultrasonography of the forearm showed an enlarged parathyroid tissue ( 17 mm  7 mm  3 mm ) .
after informed consent was obtained , we performed a simplified casanova test ( total ischaemic blockade of the graft - bearing arm ) , as previously described . following temporary ischaemic autograftectomy ,
the intact and whole pth levels significantly decreased to 127 and 117 pg / ml , respectively , with normalization of the reversed whole pth / intact pth ratio , suggesting that the autografted parathyroid tissue was the cause of the reversed ratio .
a 39-year - old female had been receiving haemodialysis since 1979 for esrd due to chronic glomerulonephritis . in 1993 ,
total parathyroidectomy with forearm autograft was performed for secondary hyperparathyroidism , which was then maintained for several years .
however , in january 2007 , elevated levels of intact pth ( 396 pg / ml ; elecsys pth ) , whole pth ( 263 pg / ml ) and bone alkaline phosphatase activity ( 63
ultrasonography of the graft - bearing arm showed an enlargement of the parathyroid tissue ( 17 mm  16 mm  5 mm ) . when pth levels were measured in the sera obtained from the graft - bearing arm , extremely elevated levels of intact and whole pth levels
were found ( 3600 and 4490 pg / ml , respectively ) , with the reversed whole pth / intact pth ratio .
after surgery , the intact and whole pth levels measured in the sera obtained from the graft - bearing arm decreased to 27 and 39 pg / ml , respectively , but without normalization of the reversed ratio .
it was then suggested that small fragments of the parathyroid tissue ( i.e. the probable cause of the reversed whole pth / intact pth ratio ) remained in the forearm . as expected , recurrent hyperparathyroidism soon developed and further treatment was required .
we report five exceptional cases with the reversed whole pth / intact pth ratio associated with progressive hyperparathyroidism , seen in relatively rapid succession in clinical practice .
three patients demonstrated an enlargement of a single hypervascular gland , and the pathological pattern and heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 suggested it to be a single nodule of uraemic hyperparathyroidism in case 1 .
the other two patients had undergone surgical parathyroidectomy and subsequently showed recurrent hyperparathyroidism due to progressive autograft hyperplasia .
abnormally higher whole pth levels than intact pth levels have been reported in a minority of patients with severe hyperparathyroidism and parathyroid cancer [ 1419 ] .
analysed serum samples from these patients by hplc and were the first to identify a new molecular form of pth with an intact n - terminal , distinct from ( 184 ) pth [ 1416 ] .
more recently , we also revealed the overproduction of n - pth in a haemodialysis patient with a single nodule of uraemic hyperparathyroidism .
this n - form of pth is detectable by the whole pth assay , but is less reactive in intact pth assays [ 1416,22 ] .
hence , the reversed whole pth / intact pth ratio strongly suggests the existence of n - pth .
this new molecular form is not produced during the peripheral metabolism of pth ( 184 ) , while we have recently shown that the source of excess n - pth is associated with the pathological parathyroid tissue . to date , our group has reported three haemodialysis patients with severe hyperparathyroidism associated with the reversed whole pth / intact pth ratio [ 1719 ] .
the first   and second   cases showed a single nodule of uraemic hyperparathyroidism for which surgical parathyroidectomy was performed , and the reversed pth ratio normalized after surgery .
the third case showed excessive growth of parathyroid gland that outstripped vascular supply , and spontaneous remission due to autoinfarction of the parathyroid gland resulted in normalization of the reversed whole pth / intact pth ratio .
the clinical features of the present cases and previously published cases with the reversed whole pth / intact pth ratio are summarized in table 1 .
all cases with the reversed whole pth / intact pth ratio showed progressive enlargement of the parathyroid gland .
these findings are in accordance with previous cases of the reversed whole pth / intact pth ratio associated with clinically worse parathyroid disease [ 1416 ] . taken together , it is suggested that overproduction of n - pth , represented by the reversed whole pth / intact pth ratio , could be an indicator of progressive hyperparathyroidism .
clinical features of haemodialysis patients with the reversed whole pth / intact pth ratio : previous and present reports hpt , hyperparathyroidism ; pr , present report .
whole and intact pth levels were measured in the sera obtained from the graft - bearing arm .
differential diagnosis between primary and secondary hyperparathyroidism is very difficult in dialysis patients with a single enlarged gland , especially those receiving haemodialysis for a long time . in this study
consequently , the advanced type of uraemic hyperparathyroidism was corroborated by the heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 , one of the genetic abnormalities responsible for tumorigenesis in primary adenoma ( case 1 ) .
thus , the reversed whole pth / intact pth ratio is also a distinct possibility in severe type of secondary hyperparathyroidism .
surgical parathyroidectomy may be required for such patients with the reversed whole pth / intact pth ratio and enlarged parathyroid gland .
recent clinical data suggest that this new form of pth has a significant biological effect on bone turnover .
the present cases also showed relatively high serum levels of bone alkaline phosphatase compared with intact pth levels . in clinical practice
, we should remember that intact pth assays usually overestimate the real activity of pth on bone , as previously shown , but they also occasionally underestimate its activity , as the present cases .
with regard to its structure , this molecular form reacts poorly in intact pth assays , having early 1218 or 1224 epitopes , but normally in the roche elecsys assay with a 2632 epitope , suggesting a modification in the region 1520 [ 1416,22 ] .
the only post - translational modification ever described is phosphorylation in the n - terminal region .
thus , several investigators postulated that n - pth could be phosphorylated on ser17 . in our patients , however , the values of the whole pth assay were abnormally higher than those of the elecsys assay with a 2632 epitope .
the precise reason for this finding is unknown , but one possible explanation is diversity in the mode of modification of this molecule . in conclusion ,
we report a series of five exceptional cases with the reversed whole pth / intact pth ratio .
it is speculated that the enlarged parathyroid gland overproduced and secreted a novel form of n - pth .
these cases suggested that the reversed whole pth / intact pth ratio or the overproduction of n - pth is associated with progressive hyperparathyroidism .
moreover , the abnormally elevated whole pth / intact pth ratio , even if it is not reversed , might indicate progressive hyperparathyroidism .
further studies are required to elucidate the clinical significance of the reversed whole pth / intact pth ratio in haemodialysis patients .
 parathyroid hormone ( pth ) levels detected by intact pth assays are generally higher than those detected by the whole pth assay because the latter does not detect non-(184 ) pth fragments , mainly pth ( 784 ) . 
 rare exceptions to this rule have been reported in patients with severe primary or secondary hyperparathyroidism and parathyroid carcinoma . 
 overproduction of an n - form of pth other than pth ( 184 ) has been observed in the sera of these patients . 
 we report five additional cases with the reversed whole pth / intact pth ratio associated with severe hyperparathyroidism in haemodialysis patients . 
 three patients demonstrated enlargement of a single hypervascular gland , whereas the other two had undergone surgical parathyroidectomy and later showed recurrent hyperparathyroidism due to progressive autograft hyperplasia . in the case of a single enlarged gland , the pathological pattern and heterogeneous expression of parathyroid adenomatosis 1/cyclin d1 suggested it to be a single nodule of uraemic hyperparathyroidism rather than sporadic primary adenoma . 
 these cases suggested that the reversed whole pth / intact pth ratio could be an indicator of marked parathyroid enlargement . 
 further studies are required to elucidate the clinical significance of the reversed whole pth / intact pth ratio in haemodialysis patients . 
Introduction
Case reports
Case 1
Case 2
Case 3
Case 4
Case 5
Discussion