Nonpalpable testes: is there a relationship between ultrasonographic and operative findings?

Background Ultrasonography (US) as a diagnostic tool in the work-up of boys with nonpalpable testes (NPT) is still controversial.
Introduction
Failure of testicular descent is a common childhood anomaly, being seen in 0.8–2% of full-term and 18–30% of premature boys. In undescended testes (UDT) the testis fails to migrate into the processus vaginalis, failing to reach the scrotum at 35 weeks of gestation. It is unilateral in 60–70% of cases. Approximately 20% of UDT are nonpalpable on physical examination [1]. There are four possible explanations for this phenomenon. First, the testis may be situated in the scrotum or inguinal canal, but is too small or there is too much subcutaneous adipose tissue. Second, the testis is located inside an open processus vaginalis in the inguinal canal and may be intermittently inguinal and abdominal in position. Third, it is in a true abdominal position; and fourth, the testis is lacking [2].
The literature reports that nearly half of viable nonpalpable testes (NPT) are in an abdominal position and 5% are in the inguinal canal. The remaining 45% are atrophic or absent, mostly as a result of in utero spermatic cord torsion and are located abdominally, inguinally or scrotally [3, 4].
Experienced paediatric surgeons can accurately diagnose UDT by repeated and meticulous palpation of the scrotal and inguinal region. Since palpation is quite subjective, the diagnosis mostly depends on the examiner’s experience and ability [2]. Currently there is controversy as to the step to take when clinical examination fails to identify a testis. Further exploration might be done by means of laparoscopy or by noninvasive methods such as ultrasonography (US), CT, MRI, venography or arteriography [3, 5, 6]. Of these, US is the imaging method of choice in children because it is noninvasive and does not use ionizing radiation. Furthermore, it is cheap and widely available [7].
When NPT is correctly diagnosed there is consensus on the need for early treatment, as this may decrease the risk of testicular cancer [8–12] and fertility problems [13–15]. We performed a study aimed at assessing the value of US in the diagnostic work-up of NPT, and to this end evaluated the relationship between preoperative US data and operative findings.
Materials and methods
During a 7-year period, 880 boys referred to the Department of Paediatric Surgery, Erasmus MC-Sophia Children’s Hospital, Rotterdam, underwent primary surgery for UDT. US was performed in 137 of these children with 156 NPT. Prior to US the patients were clinically examined by a general practitioner, a paediatrician, a urologist or, in most cases, by a paediatric surgeon. Clinical examination by a paediatric surgeon was with the boy in the supine position, the lower half of the body undressed. The examiner placed the index finger and thumb of the right hand on either side of the inguinal canal, thus preventing testes lying distally from the inguinal canal from withdrawing into the inguinal region during palpation of the scrotum. With the examiner standing on the right side of the patient, the inguinal region was then examined with the fingertips of a warm left hand. If there seemed to be no palpable testis, an attempt was made to empty the inguinal canal by carrying out an ironing movement with the fingertips stroking in the direction of the scrotum. This may reveal a palpable testis at the level of the exit of the inguinal canal, immediately shooting back deep into the inguinal canal. If no testis could be located at all, the perineum, the base of the penis and the thigh were closely examined to exclude an ectopic testis.
US was performed using an Ultramark 9 HDI unit (Advanced Technology Laboratories, Bothell, Wash.) with a high-frequency, broadband, 7–10-MHz linear array transducer or a Philips ATL HDI 5000 unit, also with a high-frequency, broadband, 5–12-MHz linear array transducer. US was performed by eight different paediatric radiologists. After US all boys were clinically re-examined by a paediatric surgeon and again on the operating table when under anaesthesia.
Subsequently, all boys underwent surgery and testicular position and other operative findings were assessed intraoperatively.
Depending on the US results for NPT different operative procedures were used. Laparoscopy was usually performed when US could not identify a testis. In the presence of a viable abdominal testis, a one- or two-stage orchiopexy was performed. Anorchia was diagnosed when a blind-ending vas deferens was identified. When the vas deferens and testicular vessels entered the internal inguinal ring the procedure was completed by an inguinal exploration. When a viable testis was found, orchiopexy was performed. When a nubbin (vanished testis) or an atrophic testis was found, this was excised for pathological examination. Inguinal exploration was usually performed when US did show an inguinal testis. When a viable testis was found, orchiopexy was performed. A viable testis was defined as having a volume according to age [16].
All US findings were compared with intraoperative findings.
Results
Eligible for this study were 137 boys with 156 NPT. Excluded from analysis were two with Müllerian inhibitory factor deficiency syndrome, implying abnormal testis position. The ages of the remaining 135 boys ranged from 4 weeks to 16.2 years. Of the remaining 152 NPT, 70 were left-sided, 48 right-sided and 17 bilateral.
Four patients (four NPT) had been referred to the radiologist by a general practitioner and 24 patients (30 NPT) by a paediatrician. Most patients, 107 with 118 NPT, were first diagnosed by a paediatric surgeon before being referred to the radiologist. All boys were seen by a paediatric surgeon after the US examination for re-examination and to discuss therapy determined by the US findings.
With knowledge of the US results, 28% of the NPT previously diagnosed as not palpable now appeared to be palpable. Three of the four NPT (75%) in patients first seen by a general practitioner were palpable when re-examined by a paediatric surgeon, as was the case for 18 of 30 NPT (60%) referred by a paediatrician. Of the NPT first examined by a paediatric surgeon, 18% were palpable on re-examination with knowledge of the US results.
US was able to locate 103 of 152 NPT (68%): 16 were found in the abdomen and 87 in an inguinal position; therefore 49 could not be found.
Comparing the US results with the surgical findings, there was a 100% positive predictive value (PPV) for the 16 abdominal testes seen on US; all were indeed found abdominally. Nevertheless, 14 viable abdominally located testes were missed by US. At surgery, all these testes were found in the lower abdomen—on the iliopsoas muscle, in the pelvis, or close to the internal inguinal ring. US located 87 inguinal testes, 84 of which were indeed found in the inguinal region (97% PPV); the other three were viable and found intraabdominally. Thirty-five of the NPT were defined as small, atrophic or vanished at operation.
Ultimately, US failed to locate 49 NPT. Nevertheless, 16 of these at surgery appeared to be viable, with 14 located intraabdominally and two in the inguinal region. Two atrophic testes were found in the abdomen. A further 22 atrophic testes were found inguinally or scrotally. Anorchia was present in nine boys.
In summary, 92 (85%) of the 108 NPTs defined as viable at operation were located by US. While nearly all viable inguinal testes (74/76) were correctly located by US, only half of the viable abdominal testes (16/33) were seen on US. The relevant data are summarized in Fig. 1.
Fig. 1Ultrasound findings in 152 nonpalpable testes in relation to findings at operative exploration
Discussion
Over the years, a wide range of investigations—from vascular techniques and imaging techniques to laparoscopy—have been used in detecting NPT [17]. Inguinal exploration used to be the standard surgical approach for nonpalpable testes. If the testis was not found at or below the internal inguinal ring, the procedure was extended to the abdomen. Laparoscopy has gained greater acceptance in diagnosing and treating NPT. Cortesi et al. [18] first reported its use in 1976 in an 18-year-old adolescent. While diagnostic laparoscopy is highly sensitive in detecting NPT [1, 2, 19, 20], it carries an approximately 1% risk of major or minor operative complications. Furthermore, the long-term incidence of peritoneal adhesions following laparoscopic procedures in children is approximately 10% [4, 21].
The literature is far from unanimous on the usefulness of US in UDT. Some authors recommend US for its feasibility and potential to settle the subsequent operative procedure [7, 21–27]. Others are sceptical because they found poor outcome for NPT, and because US does not exclude the necessity for laparoscopy [3, 28–30]. Most of the studies included only small groups of patients [24–26, 28, 30]. Not all studies investigated only NPT; some included all UDT [26, 27, 30]. Most studies compared US results with findings during the operative procedure [3, 7, 21, 22, 26–29], others compared CT and/or MRI with US [23–25, 30] in relation to the operative procedure. The most relevant conclusions of these studies are summarized in Table 1.
Table 1Review of the literature on the use of US in boys with NPT (n.a. not applicable, because all testes were ‘true’ NPT)ReferenceStudy designTestes (n)Located by USLocated “true” NPTAccuracy of US (%)aPredictive value of US (%)aRe-examinationRemarksAdvice in NPTPositiveNegativeTestes palpablePrevious examiner[22]Retrospective/medical charts5528/55 (51%)n.a.8410067n.a.–US[29]Prospective/cohort6237/62 (60%)n.a.761000n.a.Vanished not included in accuracy and predictive valuesLaparoscopy[3]Retrospective/medical charts6612/66 (18%)0/21 (0%)18b100b0b45/66 (68%)Referring physician82% US ordered by referring physician; 93% US performed elsewhereLaparoscopy[23]Retrospective/medical charts170111/170 (65%)n.a.72b100b19bn.a.–US[21]Prospective/cohort7448/74 (65%)16/29 (55%)65b100b0b45/74 (61%)Referring physicianOnly nubbins palpable on re-examinationUS[24]Prospective/cohort2113/21 (62%)n.a.769250n.a.US twice and blind to physical examinationUS → MRI[30]Retrospective/medical charts225/12 (42%)3/7 (43%)581002913/22 (59%)Referring physician–Laparoscopy[7]Prospective/cohort4545/45 (100%)6/8 (75%)9393–36/45 (80%)Referring physicianPhysical examination and US blind to each otherUS[26]Prospective/cohort3817/38 (45%)1/18 (6%)6188381/21 (5%)ExpertUS blind to physical examinationUS if obese20/41 (49%)Referring physician[28]Prospective/cohort142/14 (14%)n.a.211008n.aSurgery blind to USLaparoscopy[27]Prospective/cohort6961/69 (88%)4/12 (33%)9910088n.aUS[25]Prospective/cohort2315/23 (65%)n.a.919175n.a.US and CT blind to each otherUSaAccuracy and predictive values were (re)calculated: negative surgical exploration/absent testes were counted as true negative and testicular remnants/“nubbins” as false negative.bAbsent testes and atrophic testes grouped; number of absent testes not (exactly) mentioned in article (calculated as false negative).
An important message emerging from these articles is the very high PPV of US and the importance of thorough examination by a physician with experience in small genital examination. More than two-thirds of testes of referred patients were palpable on examination by experienced physicians. Many US investigations could have been avoided if patients had first been referred to an expert. The (paediatric) radiologist’s experience is also vital and US should be undertaken in the hospital where further treatment will be given.
US remains the modality of choice in our centre because we value its noninvasiveness, child-friendliness and cost-effectiveness. It also facilitates planning the correct surgical procedure (Fig. 2) [7, 29].
Fig. 2Flow chart representing the diagnostic and therapeutic approach in boys with NPT
In summary, we found 97% sensitivity of US for viable inguinal testes and 48% sensitivity for viable abdominal testes. When US located a testis it was also found at that site during surgery in 97% of NPT (PPV 97%). In our study, only three viable testes were not located correctly, being in the inguinal canal at US and within the abdomen at surgery. This can be explained by the fact that there was a patent internal inguinal ring with a mobile testis. More than two-thirds of NPT could be localized with US. Approximately one-third of NPT that were not found with US appeared to be viable at surgery. Of 34 testes considered nonpalpable by a general practitioner or paediatrician, 21 (62%) were palpable on re-examination by a paediatric surgeon after US. In contrast, of 118 testes considered nonpalpable by a paediatric surgeon, only 21 (18%) were palpable on re-examination.
Conclusion
We feel our results underpin the importance of referring boys with a presumed NPT to a physician with expertise in genital examination of small children. This will avoid unnecessary US investigations. Furthermore, we recommend US for all boys with NPT diagnosed by an experienced physician (Fig. 2). The findings would then determine the subsequent operation: diagnostic and/or therapeutic laparoscopy for all NPT with negative US, or intraabdominal testes located by US. In addition, we recommend inguinal exploration whenever US demonstrates an inguinal testis.


Predicting Protein Function from Structure—The Roles of Short-chain Dehydrogenase/Reductase Enzymes in Bordetella O-antigen Biosynthesis

The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid–base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function.
Introduction
The acceleration of genome sequencing has created a challenge for modern biology, namely, interpretation of the vast and growing amount of genetic information in the databases. Search algorithms such as the basic local alignment search tool (BLAST)1 can efficiently find related genes, but only in a small minority of cases is the precise function of a novel enzyme equivalent to that of the highest scoring hit. In straightforward cases the output is useful to generate hypotheses for the novel enzyme's function, based on common catalytic chemistry, substrates or both, and these postulates are then amenable to direct biochemical investigation. To make full use of genome data it is important that where such standard bioinformatic analysis cannot predict precise roles and biochemical function assays are not possible, tools are developed to differentiate the functions of genes. A good example of such a challenging case is where gene products are annotated as short-chain dehydrogenase/reductases (SDRs). Members of this family share low sequence identity (typically 15–30%) and catalyse a wide range of chemical reactions, including oxidation, reduction, epimerisation, dehydration and decarboxylation (reviewed in Ref. 2).
Bordetella
parapertussis and Bordetella bronchiseptica are pathogens of mammals. B. bronchiseptica is associated with respiratory tract infections in many animals including acute tracheobronchitis in dogs (kennel cough)3 and atrophic rhinitis in pigs,4 whereas B. parapertussis can cause whooping cough in humans.5 A separate lineage of B. parapertussis infects sheep.6
The major component of the outer leaflet of the outer membrane in Gram-negative bacteria is lipopolysaccharide (LPS). The complete structure of this macromolecule in B. bronchiseptica and B. parapertussis has been recently described.7 The lipid A domain of LPS, which consists of a diglucosamine backbone substituted with fatty acyl chains, forms the outer leaflet of the outer membranes of the bacteria. Lipid A is linked to a complex, branching oligosaccharide known as the core. Lipid A-core comprises a proportion of the LPS that is exposed on the cell surface and is known as band B. In B. bronchiseptica, lipid A-core can be substituted with a trisaccharide and this structure is known as band A. Synthesis of the band A trisaccharide requires functions encoded in the wlb gene cluster (previously named bpl).8
Both B. bronchiseptica and B. parapertussis also substitute their LPS with an O antigen. This O antigen contains 12 to 15 2,3-diacetamido-2,3-dideoxy-l-galactosaminuronic acid (l-GalNAc3NAcA) residues9 and is required for full virulence in animal and in vitro models of infection.10 O-antigen biosynthesis requires genes in the wbm cluster that is adjacent to the wlb genetic locus. The wbm locus contains three neighbouring SDR genes (wbmF, wbmG and wbmH), all of which have been annotated as nucleotide-sugar epimerases/dehydratases.
The structure of complete O antigen and the homology of wbm genes with genes of known function have led us to propose a pathway for biosynthesis of this polysaccharide and we are currently testing various steps in this scheme as part of an ongoing project to determine the functions of all 24 wbm genes. This report specifically concerns the roles of the SDR genes wbmF, wbmG and wbmH.
O-antigen residues are synthesised as sugar nucleotide precursors. The probable substrate for wbm locus-encoded biosynthesis of the nucleotide-activated l-GalNAc3NAcA is UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (UDP-d-ManNAc3NAcA). This compound is related to the l-galacto configuration by inversion of the stereochemistry at the hexose 3 and 5 positions. In the bordetellae, UDP-d-ManNAc3NAcA is formed as a precursor for band A trisaccharide synthesis by 2-epimerisation of UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid, catalysed by WlbD.11 Because there are precedents for either a single12 or multiple13–16 SDR enzymes catalysing 3,5-epimerisation conversions of sugar nucleotides, we hypothesise that one or more of WbmF, WbmG and WbmH operate in this biosynthetic pathway to catalyse the conversion of UDP-d-ManNAc3NAcA to UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid (UDP-l-GalNAc3NAcA). Because of the low percentage conversion of the WlbD-catalysed reaction, UDP-d-ManNAc3NAcA is not currently available for biochemical studies and therefore this hypothesis cannot be directly tested at the present time.
In this report we demonstrate the involvement of wbmF, wbmG and wbmH in O-antigen expression by mutation of the genes in B. bronchiseptica. Because the putative substrate is unavailable, we elected to probe the catalytic functions of WbmF, WbmG and WbmH by characterising these proteins' three-dimensional structures. With soaking and co-crystallisation experiments, densities corresponding to cofactors and nucleotide portions of substrate analogues can be identified within the active sites. Biochemical and crystallographic studies have defined the structural basis for the catalytic chemistries of a range of other SDR enzymes (reviewed in Ref. 17). Our knowledge of these structure–function relationships enables us to interpret the structures of the wbm SDRs. We have analysed the potential for acid–base chemistry in the active sites to differentiate their potential roles in vivo. Based on the composition of O antigen, the genetic context of these wbm genes and information from the structural studies, we propose roles for these genes in O-antigen biosynthesis. The results demonstrate the usefulness and limitations of X-ray data in elucidating biochemical pathways where catalytic activity cannot be directly measured.
Results
Mutational analysis of wbmF, wbmG and wbmH
To test the hypothesis that wbmF, wbmG and wbmH are involved in O-chain biosynthesis, each gene was disrupted by insertion of a tetracycline-resistance cassette. Mutation of the chromosomal genes was confirmed by Southern hybridisation (data not shown). The effects of these mutations were assessed by silver-stained SDS-PAGE analysis and immunoblotting of mutant LPS (Fig. 1). Mutation of wbmG or wbmH results in apparent abrogation of O-antigen synthesis, as the LPS from these mutants lacks O band as detected by either silver stain or Western blot. O-antigen expression in the wbmF mutant, CNF0a, is dramatically reduced compared with the wild type, but this strain retains its ability to express a small amount of material with the electrophoretic mobility of O-band LPS and which binds the O-antigen-specific monoclonal antibody, D13B11.
Each of the mutations was complemented by expression of the cognate wild-type allele in trans (Fig. 1). Each coding sequence was cloned into the broad host range vector pBBR1MCS(kan) under the control of the flaA promoter to generate complementation vectors. Complementation of the wbmG and wbmH mutations restored O-antigen expression to near wild-type levels. O-antigen expression in the complemented wbmF mutant was increased relative to the wbmF mutant, but was still considerably less than in wild type. The reason for incomplete complementation of the wbmF mutation is unknown, but is not likely to be due to polar effects of the mutation because, due to the organisation of genes in the wbm locus, wbmF is at the 3′ end of an operon.18 Introduction of the empty complementation vector had no effect on O-antigen expression in any of the mutants.
The gene products WbmF, WbmG and WbmH are all in the extended SDR family
The SDR family is divided into the classical and extended subfamilies primarily on the basis of protein length and the sequence pattern of the glycine-rich cofactor-binding region close to the N terminus. According to both criteria, the products of wbmF, wbmG and wbmH, which are predicted to have 357, 310 and 313 amino acids, respectively, and all have the GxxGxxG motif near the N terminus, are extended SDRs2 (Fig. 2).
WbmG and WbmH are closely related to each other, sharing 38% amino acid identity. WbmF, WbmG and WbmH are all homologous to Pfam01370 (NAD-dependent epimerase/dehydratase family). The most closely related, characterised homologues of these Wbm SDRs in the protein databases are deoxythymidine diphosphate (dTDP)-glucose 4,6-dehydratases from Escherichia coli and Streptomyces venezuelae with 27% or 28% sequence identity (Table 1).19 This level of sequence conservation between members of the SDR family does not imply conservation of either catalytic chemistry or substrate; rather this functional information can be better determined by examination of the residues at key positions in the active sites and binding pockets. Several conserved motifs within the extended SDR subfamily facilitate alignment of sections of the Wbm SDR sequences with homologues (Fig. 2) but many of the critical functional amino acids are located outside of these motifs, and with this low level of conservation, it is not possible to make full sequence alignments with confidence that all of these residues are correctly identified.
Crystallisation of His6-WbmF, His6-WbmG and His6-WbmH
Thus far the data implicated each of these wbm SDRs in O-antigen synthesis, but did not indicate precise roles for each gene. Therefore, in order to elucidate their individual functions we characterised the gene products by X-ray crystallography. The overexpression, purification and crystallisation of these proteins are described elsewhere.20 The structures discussed in the present work are summarised in Table 2.
General architecture of His6-WbmF, His6-WbmG and His6-WbmH
These proteins all exhibit the typical SDR family architecture (Fig. 3). The structures each comprise two domains. The first of these is the N-terminal Rossmann fold domain in which a central β sheet is flanked by two layers of α helices. The cofactor-binding motif GxxGxxG is located at the C-terminal edge of this β sheet, which has seven parallel β strands running in the order 3, 2, 1, 4, 5, 6, 7. The second domain is largely made up of C-terminal sequence and contains all of the residues involved in binding of the nucleotide portion of the substrate. The catalytic sites in SDR enzymes are located at the interface of these two domains where, in sugar-nucleotide-modifying enzymes, the substrate hexose is brought into proximity with the cofactor nicotinamide ring. The main architectural differences between these three proteins lie in their C-terminal regions (Fig. 3). The C terminus of His6-WbmG consists of a loop that stretches out from the C-terminal domain to interact with the Rossmann domain. In His6-WbmH, this loop is not visible in the density, although this difference may not reflect any distinction between the real structures of the two proteins in solution. In WbmF, the last 30 residues form a large bent helix that covers two faces of the C-terminal domain. There is also an insertion of 14 amino acids in WbmF relative to the other structures, including all residues from Gly198 to Arg212. This extra loop extends over the nicotinamide end of the cofactor-binding pocket.
SDRs typically form dimers or higher oligomers. The crystal packing of these proteins suggests that WbmF, WbmG and WbmH have the conserved four-helix bundle usually found at the dimer interface. Analytical gel filtration chromatography indicated a hydrodynamic radius for each of these proteins consistent with either a monomer or dimer (data not shown). We conclude, therefore, that these proteins are present in solution predominantly in dimer form. In the WbmG and WbmH structures, the dimer interaction buries 1316 Å2 (5.7% of total protein surface) and 1562 Å2 (7.2%) of surface per dimer, respectively. In the WbmF structure the interface region is extended to include a short antiparallel β sheet composed of the residues Ile151 to Ser153 from each monomer as well as the loops that connect these strands to the bundle (Leu154 to Pro160); 2131 Å2 is buried in the WbmF dimer interface, which is equivalent to 8.6% of the total protein surface. In all three cases, the majority of the residues buried at these interfaces are hydrophobic or aromatic.
Cofactor binding
NAD can be modelled into the electron density in all three proteins. His6-WbmG and His6-WbmH copurify with this compound from the E. coli expression host, but in order to stabilise the protein in solution His6-WbmF required the addition of exogenous cofactor,20 and this is the most likely source of this molecule in these His6-WbmF structures. The preference in each case for NAD cofactor as opposed to NADP is indicated by the close interaction (2.7 Å hydrogen bonds) of an aspartate side chain with the 2′-hydroxyl of the adenylic ribose. This side chain would disfavour binding of NADP by both steric and electrostatic repulsion of the NADP 2′-phosphate. In some SDR structures [e.g., GDP-4-keto-6-deoxymannose 3,5-epimerase/reductase (GMER)21 and RmlD22] the bound cofactor has direct access to solvent, indicating a pathway via which spent NAD/NADP can be replaced from solution. In these structures, the bound cofactor is buried within the core of the protein; in the case of the His6-WbmF structure, the 14-amino-acid insertion supplies an additional occluding loop, further blocking release of cofactor in this conformation.
Binding of substrate
Soaking and co-crystallisation experiments with His6-WbmF and His6-WbmG revealed the binding mode for the nucleotide portion of the sugar nucleotide substrates of these enzymes (Fig. 3). The X-ray data from a His6-WbmG crystal soaked with UDP-glucose revealed extra density that could be modelled as far as the α-phosphate. The location of this molecule is consistent with the nucleotide-binding site for SDR homologue structures and with the hydrogen-bonding chemistry expected to bind uridine. No density was visible corresponding to the hexose portion of the soaked compound nor the second phosphate, suggesting that either this portion of the molecule is disordered within the crystal, or the compound was partially hydrolysed and the smaller uridine monophosphate (UMP) preferentially soaked into the substrate-binding pocket. In either case, the data indicate that the glucose-1-phosphate moiety of UDP-glucose is not strongly bound in a single conformation by WbmG. A similar soaking experiment but with GDP-mannose did not indicate any binding of this sugar nucleotide (data not shown), indicating a possible preference for the uracil base. The hydrogen bonding between the WbmG pocket and UMP suggests a basis for this preference (Fig. 3b). Although we were unable to obtain any complex of WbmH with a substrate analogue bound, the nucleotide-binding domain of WbmH is conserved in sequence and structure with WbmG, implying identical specificity and a common binding mode for the nucleoside portion of their substrates (Fig. 3c). UDP density to the α-phosphate is also visible in the WbmF/NAD/UDP ternary complex. The UMP density in this structure and the chemistry of its interaction with the protein suggests that this protein, like WbmG and WbmH, recognises a UDP-sugar substrate.
Both α and β nucleotide phosphates could be modelled in the data from a UDP soak into a WbmF-NAD+ co-crystal and co-crystallisation of His6-WbmG with UDP. In both structures the β-phosphate is coordinated by a conserved arginine (Fig. 4a and b), although the precise location of this phosphate is different in the two structures and both are inconsistent with the β-phosphate positions of bound substrate in the structure of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase, DesIV, from S. venezuelae (Fig. 4c).23 In this DesIV structure [Protein Data Bank (PDB) ID 1R6D] the position of the substrate is consistent with the necessary overlap of molecular orbitals required for hydride transfer from the glucose C-4 to the cofactor. It is likely, therefore, that the β-phosphate positions in WbmF and WbmG structures do not represent the positions of equivalent phosphates when the native substrates for these enzymes are bound in the active site.
One common feature of substrate binding in SDR enzymes is pi stacking of the nucleobase with an amino acid side chain. In WbmF, this interaction involves Glu231 (Fig. 3a). Unusually, in WbmG the pi-stacking residue (Phe190) is the one that hydrogen bonds with the uracil N-3 (Fig. 3b). In WbmF, E. coli UDP-galactose 4-epimerase (GalE),24
S. venezuelae DesIV,23 and Pseudomonas aeruginosa WbpP,25 the hydrophobic contact is provided by the residue in the n + 2 position, whose backbone amide hydrogen bonds with the base's O2 group (Fig. 5). This unusual feature of the substrate binding in WbmG could not have been predicted from sequence alignments alone.
Potential catalytic chemistry
SDR enzymes have a conserved catalytic triad that is involved in their oxidoreductase activity and that is classically composed of a spatially conserved serine, tyrosine and lysine (SYK) triad, for example, Ser142, Tyr166 and Lys170 in WbpP. The first member of this triad is sometimes found as a threonine (TYK),23 and the tyrosine can be replaced by a methionine (SMK).26 The SDR triad is conserved in WbmG and WbmH as TYK but in WbmF Ala131 superimposes onto the Ser/Thr position (Fig. 6a).
In the context of O-antigen synthesis in Bordetella we are interested in the potential of the wbm SDR enzymes to catalyse secondary reactions of their substrates beyond the oxidoreductase reactions common to all SDRs. Of particular significance is the identification of a potential 3,5-epimerase. Despite the lack of a structure in which a full sugar nucleotide substrate analogue is bound, the cavity into which the substrate hexose binds is easily identifiable by comparison with homologue structures and because it is necessarily interposed between the UMP-binding site and the cofactor nicotinamide ring. The active sites of WbmG and WbmH are devoid of amino acid side chains capable of acid–base chemistry except for their respective TYK triads and, in WbmH, Ser176. SDR enzymes that catalyse 3,5-epimerisation of their substrates have a spatially conserved catalytic cysteine and a basic side chain. In GMER these residues are Cys109 and His179;21 in GDP-mannose 3,5-epimerase (GME) they are Cys145 and Lys217.12 In WbmG and WbmH these amino acids superimpose onto hydrophobic side chains, but in WbmF this catalytic cysteine is conserved (Cys133) and there are two candidates for the basic side chain, Asn213 and His90 (Fig. 6b). Cys133 is disordered in all of our WbmF crystals, indicating that an element of induced fit may be required to bring all of the active-site components into place. His90 has a very unusual backbone conformation, with a cis peptide bond to the following residue. This is strongly suggestive of a functional role for this residue, since such conformations are rarely observed except where they are required to place a key side chain in the active site.
Modelling of the putative substrate into the WbmF active site
The analysis of the potential catalytic chemistry of WbmF suggested that its role in O-antigen biosynthesis may be to catalyse the 3,5-epimerisation required in the overall conversion of UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA. The 4-keto derivative of UDP-d-ManNAc3NAcA is therefore a likely substrate or reaction intermediate for WbmF. This compound contains a bulkier sugar than any of the substrates of the characterised sugar-nucleotide-modifying SDRs. An important test of our proposed pathway is that the enzymes must be able to accommodate these unusually large substrates. We modelled 4-keto UDP-d-ManNAc3NAcA into the active site of WbmF (Fig. 7). This showed that it is feasible for the substrate to occupy the active site of WbmF in a manner that is consistent with the experimentally determined binding site for UMP, the proximity of the most likely catalytic site, and good geometry.
Discussion
The abrogation or reduction of O-antigen expression as a result of wbmF, wbmG or wbmH mutations implicates all three genes in the biosynthesis of this molecule, and is consistent with the hypothesis that these three adjacent SDR genes catalyse the UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA conversion, which is probably required for O-antigen synthesis.
Structural analysis of these three wbm SDR gene products also enables us to distinguish between them in terms of potential catalytic chemistry. Both WbmG and WbmH have the conserved SDR catalytic triad (TYK in these cases), which suggests that they should be competent to function as oxidoreductases. In both of these enzymes, the pocket that surrounds the active site is hydrophobic and featureless compared with other SDR enzymes; the lack of side chains capable of acid–base chemistry indicates that the functions of these enzymes are likely to be limited to oxidation and/or reduction and probably rules out specific 3,5-epimerase activity. The hydroxyl of Ser176 in WbmH may be capable of facilitating proton exchange; however, an amino acid pair capable of acid–base chemistry is required for 3,5-epimerase function.
Some SDRs that catalyse both oxidation and reduction bind cofactor irreversibly and recycle it within the active site.27 In other SDRs, this binding is reversible and the cofactor-binding site is situated at the bottom of a solvent-accessible groove (e.g., in E. coli GMER, PDB ID 1BWS).28 No such groove is visible in the WbmF, WbmG and WbmH structures, where the cofactor-binding sites are almost completely occluded. However, we do not take this observation as being diagnostic of irreversible cofactor binding in these proteins for the following reasons. Firstly, exogenous cofactor is required for WbmF to be stable in solution with respect to precipitation.20 Presumably either cofactor that copurifies with WbmF can be released, resulting in unfolding and aggregation of the protein, or apo-WbmF must bind cofactor supplied from the medium in order to fold correctly. Either case would suggest that NAD can be exchanged between WbmF and solvent. Secondly, human GalE reversibly binds cofactor,29 and yet does not have an open binding site like that of GMER. Finally, a conversion of configuration from d-manno to l-galacto can be catalysed by a single tight NAD+-binding enzyme such as GME,12 but we cannot envisage a pathway in which two or three such enzymes contribute to this transformation. Instead, we suggest that the protein loops that cover the NAD-binding site may be flexible and move to allow release and replacement of spent cofactor.
WbmF lacks the serine/threonine of the SDR catalytic triad. The conservation of the classical SDR catalytic triad is not absolute amongst members of the family. For example, in the very unusual SDR enzyme, biliverdin IXβ reductase (which catalyses the reduction of a range of substrates including some flavins and non-α-isomers of biliverdin), a histidine occupies the usual place of the catalytic lysine and tyrosine residues.30 The shapes and electron delocalisation exhibited by biliverdin IXβ reductase substrates imply that compared with the putative substrates of the Wbm SDR enzymes, they will have rather special requirements for binding geometry and reaction intermediate stabilisation during catalysis. In those SDRs that oxidise or reduce sugar nuclotides at the hexose C-4 position, however, the catalytic triad serine/threonine residue is important because it modifies the pKa of the C-4 hydroxyl by forming a short hydrogen bond.23 Mutation of the triad threonine (Thr134) to alanine in E. coli dTDP-Glc 4,6-dehydratase results in a 200-fold drop in kcat,31 whereas mutation of Ser124 to alanine in E. coli GalE reduces epimerase activity (kcat) almost 3000-fold.32 The incomplete WbmF catalytic triad raises doubts about the competence of this enzyme as a sugar nucleotide oxidoreductase. It is possible that the function of the missing side chain is provided by another active-site residue; this implies a different substrate-binding geometry and further studies will be required to investigate this possibility. Conversely, if a standard substrate-binding orientation is assumed, the active site of WbmF resembles that of 3,5-epimerases and our structures of this protein identify Cys133 and either Asn213 or His90 as potential mediators of proton exchange in the epimerisation reaction—one group abstracts a proton from the sugar while the other donates a proton to the opposite face.
By modelling the compound 4-keto UDP-d-ManNAc3NAcA into the WbmF structure we have demonstrated that there is space within this active site to accommodate this bulky sugar nucleotide. Other solutions produced in the modelling process demonstrated alternative plausible substrate positions (data not shown). For this reason, we do not suggest that the solution presented here (Fig. 7) represents the true active conformation. While we did not attempt similar modelling experiments with WbmG and WbmH, visual inspection of these structures indicates that they also have spacious active sites compared with other SDRs and can therefore accommodate diacetamido uronic acids in their hexose-binding pockets.
We therefore propose the following biosynthetic pathway (Fig. 8) for the 3,5-epimerisation of UDP-d-ManNAc3NAcA: One of the putative oxidoreductases, WbmG or WbmH, catalyses the initial oxidation at C-4, and the other oxidoreductase is responsible for the final reduction step. Introduction of the keto group lowers the pKa of the C-3 and C-5 hydrogen atoms, allowing WbmF to catalyse proton exchange effecting 3,5-epimerisation. This scheme is the most consistent with all of the available data. The function suggested for WbmF is very unusual in that we suggest that this enzyme does not catalyse oxidation or reduction of its substrate. If this is the case, WbmF would be the first such member of the SDR family, and this would imply that the cofactor in WbmF has a purely structural role, without participating in the catalytic cycle. It has been suggested that the transcriptional regulation protein NmrA from Aspergillus nidulans is a NAD-binding SDR family member that is not an enzyme at all. NmrA appears to function in control of nitrogen metabolism through physical interaction with the GATA family transcription factor AreA, possibly by controlling the rate of entry of AreA into the nucleus,33 and the structural equivalents of the SDR triad tyrosine and serine/threonine in NmrA are Met127 and Met113, respectively.34 Since the NmrA structure was reported, however, several SDR enzymes with SMK triads have been reported (e.g., WbpM from P. aeruginosa),26 so that the nonconservation of the SDR catalytic triad in NmrA should not rule out an enzyme function as absolutely as first appeared.34 An alternative possibility to the biosynthetic pathway we have outlined (Fig. 8) is that WbmF does in fact catalyse either oxidation or reduction as well as the 3,5-epimerisation, in which case the third SDR may participate in O-antigen expression by performing a function in the synthesis of the complicated sugar residue that adorns the nonreducing terminus of the O-chain.35 The nonredox role we propose for WbmF is consistent, however, with the lack of the conserved SDR triad in this enzyme, and if this protein operates purely as a 3,5-epimerase this may also suggest a mechanism by which the wbmF mutant is able to express O antigen, albeit in greatly reduced amounts. The product of the initial oxidation reaction is activated for proton exchange α- to the keto group and it may be that racemisation at these positions occurs at a biologically significant rate without an absolute requirement for enzyme catalysis.
Recent structural studies of the 3,5-epimerase in the dTDP-rhamnose pathway, RmlC,36 suggest that the rate-limiting step in its reaction involves a transition state in which the anomeric linkage is in the disfavoured equatorial position (which is also adopted by the reaction product), and that in order to avoid steric clashes and satisfy the requirement for axial proton abstraction, the reaction mechanism requires a twist-boat conformation such as that proposed in the GME reaction.12 Binding of substrate in a C-1 equatorial conformation may be part of the way that RmlC reduces kinetic barriers to this reaction. For the same reasons, the product of the proposed WbmF-catalysed 3,5-epimerisation will have the nucleotide attached to the l-GalNAcNAcA in the equatorial position in order to avoid 1,3,5-triaxial clashes. This transformation therefore involves considerable kinetic and thermodynamic barriers and will be slow without enzyme catalysis. However, if sufficient starting material is generated by the upstream oxidase, and the 3,5-epimerisation product is efficiently and selectively removed by the downstream reductase, the flux through this bottleneck may be sufficient to allow O-chain synthesis. Furthermore, the slower 3,5-epimerisation rate resulting from mutation of the wbmF gene may make relatively little impact on O-antigen expression levels if this conversion does not usually comprise the rate-limiting step for O-antigen synthesis. A similar effect may operate in the dTDP-l-noviose pathway in Streptomyces spheroides where observation of a naturally occurring l-rhamnoside analogue of novobiocin37 shows that 3,5-epimerisation of the dTDP-noviose precursor (dTDP-6-deoxy-d-xylo-4-hexulose) does occur despite the fact that in vitro, the 3,5-epimerisation catalysed by NovW has a 2000-fold lower kcat than RmlC38 (discussed in Ref. 39).
In conclusion, X-ray crystallography of WbmF, WbmG and WbmH enabled definition of these enzymes' NAD-cofactor preference and the substrate nucleotide specificity. We have been able to distinguish the likely function of WbmF from WbmG and WbmH, but cannot differentiate the roles of WbmG and WbmH. In order to achieve this, these proteins must be characterised in vitro. This will require chemical synthesis of UDP-d-ManNAc3NAcA and is beyond the scope of the present study. Subject to future experimental verification of predictions we have made, our data illustrate the usefulness of structural studies for investigating such challenging problems. While they also show why this methodology cannot universally supplant direct in vitro characterisation, this kind of structural analysis will prove essential for researchers to make full use of the gene databases.
Materials and Methods
Bacterial strains, plasmids and culture conditions
Bacterial strains used in this study are described in Table 3. Bordetella was grown on Bordet–Gengou agar (Difco) supplemented with 10% defibrinated horse blood (TCS Cellworks Ltd). E. coli was cultured in Luria–Bertani (LB) broth or on LB agar. All strains were incubated at 37 °C and ampicillin (100 μg ml− 1), kanamycin (50 μg ml− 1), tetracycline (10 μg ml− 1 for E. coli, 5 μg ml− 1 for B. bronchiseptica) or streptomycin (200 μg ml− 1) were added where required. Suicide plasmids were based on the host-restricted pEX100T backbone40 and broad host range shuttle vectors were based on a Kmr derivative of pBBR1MCS.41 For preparation of LPS, B. bronchiseptica was grown in medium supplemented with 50 mM MgSO4, as this maximises O-antigen expression in this strain.
DNA methods
Standard methods were used for DNA manipulations. Oligonucleotides were supplied by Sigma-Genosys. PCR was performed with template from boiled bacteria42 and Taq DNA polymerase (Promega) or KOD Hot Start DNA polymerase (Novagen).
Construction of wbmF, wbmG and wbmH mutants
The multiple cloning site (MCS) from pBluescript II SK+ (Stratagene) was excised using SacI and KpnI, blunt-ended and ligated into SmaI-cut pEX100T, to generate the MCS-containing suicide vector pEXMCS. The wbmF allelic exchange construct was prepared as follows. The wbmF gene in the plasmid Bb540g06 (a clone generated in the B. bronchiseptica RB50 genome sequencing project)43 was disrupted by insertion of a blunt-ended tetracycline-resistance (Tc) cassette into the internal NsiI site. The mutant allele was excised using Acc65I and XbaI, blunt ended, and then cloned into EcoRV-digested pEXMCS. For wbmG, the gene was amplified using primers 5′-ATATCTAGACATATGCGTATTCTGATCACCG-3′ (XbaI site underlined) and 5′-ATAAAGCTTTGATTACTGGCAACTCTTC-3′, and the PCR product was topoisomerase cloned into pCR2.1-TOPO using a TOPO-cloning kit (Invitrogen). wbmG was excised using XbaI and cloned into the XbaI site in pEXMCS. The wbmG gene was then disrupted by cloning a blunt-ended Tc cassette into the internal Acc65I site. The wbmH mutant allele was obtained by in vitro transposon-mediated mutagenesis of the wbm locus-containing cosmid, BbLPS118 (accession number AJ007747) using an EZ-Tn5™ <Tet-1> insertion kit (Epicentre). The <Tet-1> transposon, plus flanking wbmH DNA, was cut out by partial digestion with AluI and ligated into SmaI-cut pEX100T. Allelic exchange constructs were electroporated into E. coli SM10λpir and transferred to B. bronchiseptica by conjugation with E. coli SM10λpir as donor.44 Loss of the plasmid-encoded sacB gene in allelic exchange mutagenesis of B. bronchiseptica was selected for by growth on LB agar with reduced salt concentration and supplemented with 10% (w/v) sucrose.45
Complementation of mutations
The B. bronchiseptica
flaA promoter was amplified using primers 5′-GCTCTAGATAGGCGCATGCCATGGCC-3′ (XbaI site underlined) and 5′-AAGGATCCCATATGGAGGCTCCCAAGAGAGAA-3′ (BamHI and NdeI sites underlined), and cloned into the XbaI and BamHI sites in pBBR1MCS-kan to generate the vector pCompEmpty. wbmF was amplified using primers 5′-AAAAAAACATATGTTGCCAGTAATCATGAATGC-3′ (NdeI site underlined) and 5′-AAAAAGCTTAGATCTAGTCCGCCGTCTTATTTG-3′ (HindIII site underlined) and cloned into pCompEmpty using NdeI and HindIII to generate the wbmF complementation vector pCompF. wbmG was PCR amplified using primers 5′-AAAAAAACATATGCGTATTCTGATCACC-3′ and 5′-AAAAAGCTTAGATCTGCAACTCTTCAGGTCTTG-3′ and cloned into pCompEmpty using NdeI and HindIII, generating pCompG. wbmH was amplified using 5′-GAGAATTCCATATGAAGAAAGTATTTATTACGG-3′ and 5′-AAAAAGCTTAGATCTTTGTCGATGACCAGGATT-3′ and cloned into pCompEmpty using NdeI and HindIII, generating pCompH. Shuttle vectors were moved into B. bronchiseptica by conjugation with E. coli CC118 as donor46 and trans-acting transfer functions provided by E. coli S17-1 pNJ5000 as helper.44,47
SDS-PAGE, silver stain and immunoblot analysis of LPS
LPS was obtained from B. bronchiseptica using a modification of the method of Hitchcock and Brown48 as has been described.49 SDS-PAGE of LPS was performed using Novex precast 16% tricine gels (Invitrogen). LPS was oxidised in-gel with periodic acid50 and visualised with the Silver Stain Plus kit (BioRad). Western blotting was performed as previously reported51 using the monoclonal antibody D13B11 that specifically binds the O-antigen-containing LPS of B. bronchiseptica strain CN7635E.35
X-ray crystallography
Protein production, purification, crystallisation and data collection were performed as described.20 Data were processed using DENZO and SCALEPACK (version 1.97).52 Data were truncated and converted to structure factors using TRUNCATE53 from the CCP4 Suite.54 Molecular replacement was carried out using PHASER55 as described.20 For the initial WbmG structure, phases were determined experimentally from selenomethionine-labelled protein: peak, inflection point and high-energy remote data were used to determine initial phases using the PHENIX package.56 Model building was carried out using COOT,57 and refinement was performed with REFMAC version 5.0.58 Initial models were improved using ARP/wARP version 6.1 where the data quality permitted.59 The CCP4i interface60 was used where appropriate. TLS parameters were assigned (where used) using the TLSMD server.61 CNS62 was used to perform simulated annealing. Structures were validated using COOT, PROCHECK,63 WHATCHECK,64 and RAMPAGE.65
Table 2 provides a summary of the crystallographic statistics. Protein structures were analysed using Pymol,66 COOT,57 and CCP4MG.67 Surface area calculations were performed using the Richards' Rolling Probe method.68 More extensive details of the crystallographic methods are provided in the Supplementary Data.
Modelling of putative substrate into WbmF structure
Coordinates and parameters for the 4-keto derivative of UDP-2,3-dideoxy-2,3-diacetamido-d-mannuronic acid were prepared using the CCP4 monomer library sketcher with refinement in REFMAC 5.2.58 An initial structure was prepared by modelling the proposed substrate into the structure of WbmF co-crystallised with UDP in COOT.57 The observed density was used to place the atoms present in UMP, and to place sugar atoms in positions occupied by water or glycerol in the observed structure. Different orientations were used in the two molecules in the asymmetric unit to increase the sampling of conformational space. The structure of WbmF co-crystallised with UDP, with 4-keto UDP-d-ManNAc3NAcA placed in, was truncated to include only protein, NAD and 4-keto UDP-d-ManNAc3NAcA atoms. This was used as an input to MODELLER version 9.2 to determine an optimised structure for the complex with the sugar nucleotide.69 After modelling, the ligand coordinates were regularised in COOT to correct minor distortions. Ten modelling trials were performed with different seeds, and the best was selected by eye on the basis of geometry, agreement with the determined structures and lack of bad interactions.
Protein Data Bank accession codes
X-ray amplitudes, phases and the derived atomic coordinates have been deposited with the PDB under the accession codes 2PZJ, 2Q1T, 2Q1U, 2Q1S, 2PZK, 2PZL, 2PZM and 2Q1W.

blast, basic local alignment search tool


Inter-scan reproducibility of coronary calcium measurement using Multi Detector-Row Computed Tomography (MDCT)

Purpose To assess inter-scan reproducibility of coronary calcium measurements obtained from Multi Detector-Row CT (MDCT) images and to evaluate whether this reproducibility is affected by different measurement protocols, slice thickness, cardiovascular risk factors and/or technical variables.
Introduction
A considerable proportion of the western society is at risk of suffering a cardiovascular event during life. Atherosclerosis is one of the main underlying processes. Non-invasive assessment of atherosclerosis is important since it allows studies into the etiology and consequences of early and advanced atherosclerosis in populations at large [1]. The last two decades, measurement of coronary artery calcification (CAC) using computer tomography (CT) has been used to assess coronary atherosclerosis non-invasively. The presence, and more importantly, the quantity of CAC, relates well with the overall severity of the atherosclerotic process [2]. Several studies have demonstrated a strong relation between coronary calcium burden and the incidence of myocardial infarction, a relation which was independent of age [3, 4].
Most of the evidence on determinants and consequences of coronary calcium is based on data obtained with electron beam CT (EBCT) [5–7]. The availability of EBCT scanners is modest, whereas the Multi Detector-Row CT (MDCT) scanners are more widely available and also allow for detection of coronary calcium. Current data suggest that EBCT and MDCT give comparable results [8, 9]. In contrast to EBCT, however, data on reproducibility of CAC measurements using MDCT images is not widely available [10, 11], but information is relevant. Furthermore, due to technical improvement, slice thickness of the images has become smaller which may affect the likelihood of detecting coronary calcium, and hence its reproducibility.
We set out to study inter-scan reproducibility of coronary calcium measurements from MDCT images and to evaluate whether reproducibility is affected by different measurement protocols, slice thickness, selected cardiovascular risk factors and technical variables.
Materials and methods
Participants were recruited from the PROSPECT study [12], cohort of 17,357 healthy breast-cancer screening participants, aged 49–70 years, living in Utrecht and surroundings, enrolled between 1993 and 1997. Between October 2002 and December 2004, a random selection of 1,996 women were invited by mail and 1,000 (50.1%) who were postmenopausal and did not use contraceptives or hormone replacement therapy answered positively. Of these 1,000 women, a random selection of 573 underwent a MDCT examination during a single visit and 76 of them were scanned twice. The Medical Ethical Committee of the University Medical Center Utrecht approved the study and written informed consent was obtained from all participants.
Current cardiovascular drug use (blood pressure lowering, lipid lowering and glucose lowering drugs) was assessed by asking women to bring all packages to the study centre. Smoking behavior, medical history and cardiovascular family history were assessed by a questionnaire. Height and weight were measured and body mass index was calculated as weight divided by height squared (kg/m2). Waist-to-hip ratio (WHR) was assessed. Systolic and diastolic blood pressures were measured at both arms with an automated and calibrated blood pressure device (DINAMAPTM XL, Critikon, Johnson & Johnson, Tampa, Florida, USA) with the subject in supine position. A venous blood sample was drawn after an overnight fast of at least eight hours. Plasma total cholesterol, plasma triglycerides, and plasma glucose were measured using standard enzymatic procedures. High-density lipoprotein (HDL) cholesterol was measured by the direct method (inhibition, enzymatic). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula.
Coronary imaging and calcium measurements
The amount of calcium in the coronary arteries was assessed with a Multi Detector-Row CT (MDCT) scanner (Mx 8000 IDT 16, Philips Medical Systems, Best, The Netherlands). Subjects were positioned within the gantry of the MDCT scanner in supine position. During a single breath hold, images of the heart, from the level of the tracheal bifurcation to below the base of the heart, were acquired using prospective ECG triggering at 50–80% of the RR-interval, depending on the heart rate. Scan parameters were 16 × 1.5 mm collimation, 205 mm field of view (FOV), 0.42 s rotation time, 0.28 s scan time per table position, 120 kVp and 40–70 mAs (patient weight <70 kg: 40 mAs; 70–90 kg: 55 mAs; >90 kg: 70 mAs). Scan duration was approximately 10 s, depending on heart rate and patient size. We had the participant get up from the table and lay down again since in studies on change in CAC over one year it is not realistic to assume exactly the same position of the participant at both occasions. Therefore our patients sat up and consequently moved slightly between scans to mimic two separate scan runs.
From the acquired raw data, the whole volume was reconstructed with an intermediate reconstruction algorithm in non-overlapping data sets of 1.5 mm and 3 mm thick sections. Quantification of coronary calcium was performed on a separate workstation with software for calcium scoring (Heartbeat-CS, EBW, Philips Medical Systems, Best, The Netherlands). All regions with a density over 130 Hounsfield units were identified as potential calcifications.
After completing a training-program, one scan reader (AR) who was unaware of the scores of the first scan, manually selected the calcifications within one of the coronary arteries (left main, left anterior descending, left circumflex, right coronary artery, and PDA) and scored the second scan of the participants. To reduce the influence of noise, the minimum size of a calcified lesion was set at 0.5 mm2. The peak density in Hounsfield units and the area in mm2 of each selected region were calculated. The Agatston [13] calcium score was obtained by multiplying the area by a weighting factor that is dependent on the peak signal anywhere in the lesion. The scores of individual lesions were added to obtain the Agatston calcium score for the entire coronary tree. The total calcium volume was calculated by multiplying the area of the calcified lesion (measured in square millimeters) by section thickness (1.5 mm and 3.0 mm). The calcium volume for each coronary vessel was computed by summing the volumes of the lesions in that vessel for all sections. Finally, the total volume from all the vessels became the calcium volume for a subject. The mass method uses volumetric, density information and a calibration phantom of hydroxyapatite to calculate the actual mass of the calcified plaques [14].
In addition, information on breathing artifact (inconsistency of sternum bone in sagital section in mm), noise (standard deviation of enhancement in fixed cardiac area of 212 mm2) and mean heart rate (beats/min) during scan acquisition was collected.
Data analysis
The mean and standard deviations (SD) of coronary calcium were calculated for all scoring methods separately. Because of the skewed distribution of scores, medians were also computed. The Intra-class correlation coefficient was estimated for between scans data and for 1.5 and 3.0 mm slices thicknesses separately. The mean difference in score between scans was calculated as well as the absolute and relative differences.
To distinguish between random differences or systematic difference, information on mean and absolute differences is needed. One may assume a priori a non-differential misclassification in the calcium scores, but one has to show that with the results. When the chance of the 2nd result being higher or lower is equal, one would expect a mean difference of zero, with some standard deviation. The absolute difference will not be zero since all differences are ‘absolutised’, but it is expected that at least the mean difference is much less than the absolute difference. If however the chance of a higher or lower value in the 2nd scan is not equal, the mean difference will be plus or minus a certain value. In addition, the absolute difference will have a value close to that of the mean difference. Therefore we need both parameters.
To estimate a weighted kappa as measure of agreement of categorical variables, subjects were divided into four groups according to the mean Agatston score as proposed by Rumberger et al. [15]: A: 0–9 (absent-minimal), B: 10–99 (mild), C: 100–399 (moderate) and D: (400 (severe degree of calcification). This categorization is specifically for the calcium scoring method according to Agatston. Therefore we additionally categorized all scoring methods in their quartiles to calculate kappa as measure of agreement for all scoring methods.
The relation between risk factors, technical variables and measurement error was assessed using Spearman correlation coefficients. In a similar manner the relation between calcium level and measurement error was examined. Since logarithms of coronary calcium scores have generally been used in statistical analyses in other papers, we also studied the reproducibility of logarithmic transformed calcium score. Logarithmic analysis of coronary calcium scores was performed by calculating natural log of coronary calcium scores +0.001 (ln (CCS + 0.001)) because the logarithm of coronary calcium scores alone excludes all subjects with zero scores [16]. We defined relative difference as absolute difference divided by the mean calcium level multiplied in 100 and expressed in percent. Data analysis was performed with SPSS for windows, version 12.0. A statistically significant difference was assumed when the two-sided P-value was less than 0.05.
Results
Mean age was 67.3 ± 5.2 years. Fifty-five participants (72.4%) had a coronary calcification more than zero in Agatston (1.5 mm slice thickness). Table 1 shows the general characteristics of the 76 women who had two MDCT scans.
Table 1Characteristics of studied population (N = 76)MeanStd. deviationAge (year)67.35.2BMI (Kg/m2)26.33.9WHR0.840.06SBP (mmHg)133.918.9DBP (mmHg)71.79.1Total cholesterol (mmol/l)6.090.86LDL cholesterol (mmol/l)4.310.97HDL cholesterol (mmol/l)1.510.36Triglycerides (mmol/l)1.280.62Glucose (mmol/l) 4.050.69Heart rate (beat/minute)7211Current smoking (%)*11Former smoking (%)43Previous CVD (%)1Family history of CAD in either parents (%)10BMI = Body Mass Index; CAD = Coronary Artery Diseases; DBP = Diastolic Blood Pressure; LDL = Low Density Lipoprotein; HDL = High Density Lipoprotein; SBP = Systolic Blood Pressure; WHR = Waist to Hip Ratio* Percentages have been rounded
Table 2 presents information on calcium distributions by various scoring techniques and reproducibility results, by slice thickness. Overall, calcium scores were higher when based on the 1.5 mm slice thickness than based on the 3.0 mm slice thicknesses. The kappa agreement and Intra-class correlation coefficients between the two scans were high for all scoring methods, indicating that with respect to ranking of subjects all three methods are doing well. In addition, the mean differences in scores were relatively small compared to the absolute differences for all measurements, suggestion no systematic measurement errors. Finally, results for the scans with 1.5 mm slice thickness were similar to those for the 3.0 mm slice thickness (Table 2).
Table 2Characteristics of different coronary calcium scoring methods; effect of slice thickness on inter-scan reproducibilityMass 1st ScanMass 2nd Scan Volume 1st Scan Volume 2nd Scan Agatston 1st Scan Agatston 2nd Scan Slice thickness 1.5 mmMean32.2131.88154.52149.40170.33163.63Median6.156.0539.9736.5231.8532.00Agreement (k) Rumberger categories0.970.890.87Agreement (k) Quartiles0.840.810.88Mean difference0.35.16.7Absolute difference4.022.324.3Relative difference (%)12.414.614.5ICCC*0.990.990.98Slice thickness 3.0 mmMean25.5725.45131.45126.98140.06135.82Median4.003.6530.3021.9020.3018.00Agreement (k) Rumberger categories0.920.830.73Agreement (k) Quartiles0.840.840.84Mean difference0.14.44.2Absolute difference3.518.721.3Relative difference (%)13.714.715.4ICCC*0.990.980.98* Intra-class correlation coefficient
Table 3 presents the relation of cardiovascular risk factors with inter-scan mean difference. No consistent relations were found between risk factor levels and measurement error. Importantly, however was the observation that calcium level or the logarithm of the coronary calcium scores were not related to the mean difference between scans, whereas they were significantly related to the absolute and relative differences (Table 4, Figs. 1 and 2). These observations suggest that measurement error increases with increasing CAC levels, yet that this occurs in a random way.
Table 3Relationship between cardiovascular risk factors and inter-scan mean difference of coronary calcium scoring methods by MDCT (Slice thickness 1.5 mm)Inter-scan mean differenceCCS methodsMassVolumeAgatstonBiological variablesrP-valuerP-valuerP-valueBMI (Kg/m2)0.040.730.030.740.020.80Age (year)0.180.100.310.000.280.01Smoking(Categorical)−0.000.980.040.710.070.49WHR−0.030.730.080.480.130.24SBP (mmHg)0.100.370.160.140.240.03DBP (mmHg)0.160.140.050.610.110.34Cholesterol (mmol/l)−0.270.05−0.120.40−0.200.17LDL (mmol/l)−0.180.10−0.190.09−0.090.40HDL (mmol/l)−0.040.72−0.160.14−0.110.34Triglyceride (mmol/l)−0.020.850.130.240.110.34Glucose (mmol/l)0.160.24−0.000.980.000.98Mean heart rate−0.030.77−0.030.73−0.020.81Technical variablesMean breathing artifact0.010.88−0.030.78−0.020.87Mean SD of noise0.130.260.080.490.070.52Coronary calciumMean mass score0.000.98Mean volume score0.030.75Mean Agatston score0.020.86Mean log mass score0.000.99Mean log volume score0.030.76Mean log Agatston score0.020.85BMI = Body Mass Index; DBP = Diastolic Blood Pressure; LDL = Low Density Lipoprotein; HDL = High Density Lipoprotein; r = spearman correlation coefficient; SBP = Systolic Blood Pressure; WHR = Waist to Hip RatioTable 4Relationship between cardiovascular risk factors and inter-scan absolute and relative difference of coronary calcium scoring methods by MDCT (Slice thickness 1.5 mm)Inter-scan relative differenceCCS methodsMassVolumeAgatstonBiological variablesrP-valuerP-valuerP-valueBMI (Kg/m2)0.070.530.080.460.090.43Age (year)0.210.060.240.030.l170.12Smoking(Categorical)−0.030.73−0.070.51-0.140.20WHR0.070.550.050.660.050.66SBP (mmHg)0.060.570.040.680.110.32DBP (mmHg)0.320.0040.310.0050.330.003Cholesterol (mmol/l)0.130.370.100.500.001.00LDL (mmol/l)−0.140.21−0.170.12-0.180.11HDL (mmol/l)0.070.520.040.670.060.57Triglyceride (mmol/l)0.030.780.070.490.000.99Glucose (mmol/l)0.230.090.260.050.240.08Mean heart rate−0.010.910.010.930.000.97Technical variablesMean breathing artifact0.100.440.090.490.150.23Mean SD of noise0.190.090.190.090.180.11Coronary calciumMean mass score0.290.009Mean volume score0.330.003Mean Agatston score0.380.001Mean log mass score0.290.010Mean log volume score0.330.003Mean log Agatston score0.370.001Inter-scan absolute differenceTechnical variablesMean breathing artifact0.120.320.120.330.150.22Mean SD of noise0.200.080.190.090.150.17Coronary calciumMean mass score0.86<0.001Mean volume score0.84<0.001Mean Agatston score0.89<0.001Mean log mass score0.86<0.001Mean log volume score0.83<0.001Mean log Agatston score0.89<0.001BMI = Body Mass Index; DBP = Diastolic Blood Pressure; LDL = Low Density Lipoprotein; HDL = High Density Lipoprotein; r = spearman correlation coefficient; SBP = Systolic Blood Pressure; WHR = Waist to Hip RatioFig. 1Relation between mean calcium score and inter-scan difference in mean calcium scores (Bland-Altman plots)Fig. 2Relation between mean calcium score and inter-scan absolute difference
Discussion
With respect to ranking of subjects, the inter-scan reproducibility of coronary calcium measurements by MDCT using Agatston, volume and mass scoring algorithms is excellent. The inter-scan reproducibility showed no major differences between scoring methods. The slice thickness did not affect reproducibility, nor did heart rate and technical parameters. Measurement error was related to increased coronary artery calcification, although our findings suggest that the error in the measurements is a random phenomenon.
Our findings, i.e., no major differences between scoring methods are in contrast with several reports on reproducibility based on EBCT scanning. Direct comparison of the findings of these studies with those of other is difficult since the parameters used to indicate reproducibility differ between studies. Furthermore, potentially the prevalence of CAC and its extent may affect reproducibility, as our findings suggest that measurement error increases with increasing CAC levels. Also the sizes of the studies differ which have undeniable effects on reproducibility results. However, our results are similar to those of by Rumberger and Kaufman [17], who compared these three methods and did not find any one method preferable to another in terms of reproducibility of results from consecutive scans in a patient.
Although the correlation between inter-scan measurements is excellent [18, 19], it still occurs that subjects with small deposits of calcium in scan one may have larger deposits of calcium in the 2nd scan, which leads to proportionally larger error in reproducibility. This has triggered other studies [20] on reproducibility to suggest that “the variability is partially a function of the absolute calcium score and inversely related to it”, implicating that low coronary calcium scores may not be reproducible. However, our results could not confirm this.
Besides different algorithms for calcium scoring, slice thickness has been reported to affect the reproducibility of scoring protocols. In our study, the reproducibility of the coronary calcium measurements by MDCT was similar for 1.5 mm as for 3.0 mm slice thickness, and equal for Agatston, volume and mass measurements confirming the results by Rumberger and Kaufman [17].
The implications of our main findings depend on the research question that is asked in studies using CAC measurements. When the interest is using CAC measurements for prognostic studies our results for kappa and ICCC show that ranking of subjects is adequate based on one CT scan. So the need for duplicate CAC scan is absent. The fact that measurement error increases with increasing CAC values, is in prognostic studies not of major importance since the categorization of individuals seems adequate. When the interest is in etiologic studies using CAC as outcome parameter, our findings show that risk factor relations will be validly estimated since none of the risk factors relates to measurement error. When the interest is in using CAC as risk factor for future events (assessment of relative risks), it is most likely that in analyses with CAC as continues variable the magnitude of association of high CAC levels with events reflects an underestimation of the true magnitude. The direction of the relation will not change since based on our results measurement error is random, leading to random misclassification of the exposure variable. When the interest is in diagnostic value of CAC measurements, which is usually done in categories of CAC, again the relations will be valid given our high kappa coefficients. Although our study was performed in healthy postmenopausal women, we expect that the finding will also be applicable for men.
Our findings are important in the light of the wider availability of MDCT in countries compared to EBCT. One reason for that is lower equipment cost. Other advantages of MDCT over EBCT have been suggested to be less quantum noise, thinner section thickness, and simultaneous acquisition of four sections (with 16-slice or with 64-slice ), which is reported to reduce misregistration artifact.
In conclusion, our findings demonstrate that coronary calcium measurements by MDCT are highly reproducible and are not affected by scoring protocols, slice thicknesses and technical factors.

reproducibility
multi detector-row ct (mdct)
atherosclerosis
coronary artery calcification
epidemiology