Low anterior resection syndrome following rectal cancer surgery: are incidence and severity lower with long-term follow-up?

BACKGROUND: Low anterior resection syndrome (LARS) is a functional disorder that may follow restorative proctectomy. The aim of this study was to evaluate the long-term incidence and risk factors for LARS following surgery for rectal cancer. METHODS: A retrospective study was performed on patients from a prospectively maintained database, who underwent a restorative proctectomy between January 2014 and December 2019 at Hadassah Hebrew University Medical Center. The study cohort was divided into two groups: patients following partial proctectomy with a partial mesorectal excision and a colorectal anastomosis (PME group) and patients following total proctectomy with total mesorectal excision and a coloanal anastomosis (TME group). The incidence and severity of LARS were evaluated using the LARS questionnaire. Risk factors for LARS were also evaluated. RESULTS: A total of 240 patients (male: female ratio 134:106, median age 64 years [interquartile range 55-71 years]) were included in the analysis. There were 160 patients in the PME group and 80 patients in the TME group. The overall incidence of LARS was 37.4% (26.5% major LARS and 10.9% minor LARS). Major LARS was observed in 53.8% of patients who underwent TME and in 12.7% of patients who underwent PME (p < 0.001). On multivariate analysis, distance of the anastomosis from the anal verge, TME, and neoadjuvant radiotherapy were independent prognostic factors for LARS. The incidence and severity of LARS were significantly lower in patients with longer follow-up (p = 0.05). CONCLUSIONS: Significant improvement in LARS can be expected with longer follow-up. Distance of the anastomosis from the anal verge, TME and neoadjuvant radiotherapy are independent risk factors for LARS.

External sphincter-sparing anal fistulotomy (ESSAF): a simplified technique for the treatment of fistula-in-ano.

BACKGROUND: Fistula-in-ano due to cryptoglandular disease is a common condition. While a simple anal fistula can be treated successfully by a fistulotomy, the risk of potential damage to the anal sphincters and subsequent poor functional outcomes persist in a large portion of patients with complex fistulae. Several sphincter-preserving treatment procedures have been described for complex fistulae over the past 3 decades, with variable results and complication rates, and no procedure is proven to be superior to the others. We developed external sphincter-sparing anal fistulotomy (ESSAF), a reproducible simple modification of the ligation of intersphincteric fistula tract (LIFT) technique for the treatment of complex fistula-in-ano.. The aim of the present study was to describe the technique and our outcomes. METHODS: This was a retrospective review of all patients who underwent ESSAF for a complex anal fistula at our institution from January 2014 to December 2019. The primary outcome measure of this study was the primary fistula healing rate. Secondary outcome measures included fecal and/or gas incontinence and postoperative complications. During the ESSAF procedure, the mucosa and skin overlying the fistula tract are incised to allow complete exposure of the sphincter complex. Then the internal sphincter muscle fibers overlying the tract are divided and the tract is meticulously curetted and debrided. Next, the internal opening of the tract traversing the external sphincter muscle is suture-ligated with absorbable sutures. Then, a minimal amount of mucosa is advanced and the incision is partially closed with absorbable sutures, while its external portion is left open for drainage. RESULTS: Fifty-nine patients [43 males, median age was 50 years (range 36-63 years)] underwent ESSAF for complex anal fistula during the study period. Mean follow-up was 12 ± 14.7 months. Of the 59 patients, 42 (71.2%) experienced fistula closure, with a median healing time of 8 weeks (IQR 4-16 weeks). None of the patients developed significant anal incontinence following the procedure. One patient (1.7%) suffered from soiling and another patient (1.7%) developed postoperative bleeding. There were no infectious complications. Of the 17 patients (28.8%) who failed to heal successfully, 9 (15.2%) did not heal primarily and 8 (16%) experienced recurrence after complete healing. Thirteen (76%) of these patients underwent reoperation with complete recovery after ESSAF (n = 4), fistulotomy (n = 8) or endorectal advancement flap (ERAF) (n = 1). Overall ESSAF initiated recovery in 93.2% of the patients. CONCLUSIONS: ESSAF is a feasible, safe, reproducible and effective sphincter-sparing procedure for the treatment of complex anal fistulae.

Does caffeine enhance bowel recovery after elective colorectal resection? A prospective double-blinded randomized clinical trial.

BACKGROUND: Postoperative ileus is a common condition following abdominal surgery. Previous studies have shown the positive effects of coffee on gastrointestinal motility. The aim of this study was to assess whether caffeine is the stimulatory agent in coffee that triggers bowel motility and thus may reduce the duration of postoperative ileus. METHODS: This was a single-centered, prospective, randomized controlled, double-blinded clinical trial. Patients scheduled to undergo elective laparoscopic colectomy between November 2017 and March 2019 were randomly assigned to receive either oral caffeine (100 mg three times daily) or placebo following the procedure. Primary endpoints were time to first flatus and time to first bowel movement. Secondary endpoints were time to tolerate a solid, low-residue diet and length of hospital stay. Registration number: NCT03097900. RESULTS: Seventy patients were included, [35 males, median age 56 years (range 19-79 years)]. After the exclusion of 12 patients, there were 30 patients in the caffeine group and 28 patients in the placebo group. The first passage of stool in the caffeine group occurred 18 h earlier than in the placebo group (p = 0.012); other endpoints did not reach statistical significance. No caffeine-related adverse events were observed. CONCLUSION: Caffeine consumption following colectomy is safe, leads to a significantly shorter time to first bowel movement, and may thus potentially lead to a shorter postoperative hospital stay.

Transanal total mesorectal excision for rectal cancer with indocyanine green fluorescence angiography.

BACKGROUND: The aim of this study was to evaluate the impact of fluorescence angiography (FA) on any change in proximal resection margin and/or anastomotic leak (AL) following transanal total mesorectal excision (TaTME) for rectal cancer (RC). METHODS: This retrospective cohort study was conducted at two centers by three senior surgeons. Both institutions' prospectively maintained Institutional Review Board-approved databases were retrospectively queried for all consecutive patients between July 2015 and May 2017 who had laparoscopic hybrid trans-abdominal total mesorectal excision (TME) and TaTME for RC with colorectal or coloanal anastomosis < 10 cm from the anal verge. All patients had intraoperative FA to assess colonic perfusion of the planned proximal resection margin before bowel transection and after construction of the anastomosis. Primary outcomes measured any changes in proximal resection margins and AL rates. RESULTS: Fifty-four patients (31 males; mean age 63 ± 12 years) were included; 30 (55%) of whom received neoadjuvant chemoradiation. The average anastomotic height was 3.6 cm from the anal verge and 8 (14.5%) patients required intersphincteric dissection. Forty-six patients (85%) had loop ileostomy. FA led to a change in the proximal resection margin in 10 patients (18.5%), one of whom had AL on postoperative day 3 requiring diagnostic laparoscopy and loop ileostomy. A second patient, without a change in the proximal resection margin, also had an AL. The overall AL rate was 3.7%. CONCLUSIONS: FA changed the planned proximal resection margin in 18.5% of patients, possibly accounting for the relatively low AL rate. FA is imperfect, and subjective but does have the potential to improve outcomes.

Review: The compositional variation of the rumen microbiome and its effect on host performance and methane emission.

The rumen microbiome has the important task of supplying ruminants with most of their dietary requirements and is responsible for up to 90% of their metabolic needs. This tremendous feat is possible due to the large diversity of microorganisms in the rumen. The rumen is considered one of the most diverse ecosystems on the planet in terms of species diversity and functional richness. From the moment the feed is ingested, it enters a vast cascade in which specialized microorganisms degrade specific components of the feed turning them into molecules, which in turn are utilized as anabolic precursors and energy sources for the animal. The output of this degradation process not only affects the animal, but also has an extensive impact on the environment. Some of the byproducts that are emitted as waste from this process, such as methane, act as greenhouse gases which greatly contribute to global warming. Recent technological advances developed to study this community enabled a larger overview of its vast taxonomic and functional diversity, thus leading to a better understanding of its ecology and function. This deeper understanding of the forces affecting the microbiome includes the forces that shape composition, the variation among animals, the stability of its key components, the processes of succession on a short- and long-time scales such as primary colonization and diurnal oscillations. These collective understandings have helped to provide insights into the potential effects that these forces have on the outputs observed from the animal itself. Over the recent years, there has been a growing body of evidence demonstrating the link between the microbiome and its effect on productivity of the host animals and the environment, which has placed rumen microbiome studies in the forefront of animal agricultural research. In this review, we focus on the natural variations in community composition, which are not the results of different management or feed but rather intrinsic features of animals. We characterize the rumen microbiome, its potential impact on its host as well as the barriers in implementing the current knowledge to modulate the microbiome and point toward potential avenues to overcome these hurdles.

Indocyanine green fluorescence angiography during low anterior resection for low rectal cancer: results of a comparative cohort study.

BACKGROUND: Anastomotic leak (AL) after low anterior resection (LAR) is associated with increased morbidity, mortality, cost and cancer recurrence rates. The aim of this study was to evaluate the impact of fluorescence angiography (FA) on AL following LAR for low rectal cancer. METHODS: This is a single surgeon retrospective cohort study with a historical, consecutively sampled case matched control group. The institution's prospectively maintained institutional review board (IRB)-approved database was queried for all patients who underwent a laparoscopic LAR for rectal neoplasia with a colorectal or coloanal anastomosis < 5 cm from the anal verge between 2013 and 2016. Patients were divided into two groups: patients in whom FA was employed (study group, 2015-2016) and those patients in whom it was not (control group, 2013-2015). All patients were diverted with a loop ileostomy. The primary outcome measured was the AL rate and the secondary outcome measured was change in surgical plan following FA. RESULTS: Sixty patients were included in the study: 30 patients in the FA group and 30 patients in the control group. Patients' demographics, the use of neoadjuvant chemoradiation, tumor stage, and mean height of anastomosis were comparable between the study groups. FA led to a change in surgical plan in four patients (13.3%) none of who suffered an AL. Two patients in the control group had a clinically and radiologically confirmed AL, whereas there were no leaks in the FA group (6.7% vs. 0%, p = 0.49). CONCLUSIONS: FA changed the surgical plan in 13.3% of LAR's, potentially reducing the incidence of AL in these high-risk patients.

Effect of feeding long or short wheat hay v. wheat silage in the ration of lactating cows on intake, milk production and digestibility.

The objective of this study was to evaluate in lactating cows the effect of either chopping or ensiling of wheat roughage on: intake, digestibility, lactation performance and animal behavior. Three groups of 14 lactating cows each, were fed total mixed rations (TMRs) based on either long wheat hay (HL), short wheat hay (HS) or wheat silage (SI), as the sole roughage source (30% of TMR dry matter (DM)). Parameters examined: sorting behavior, DM intake, milk yield and composition, rumination, recumbence, average daily rumen pH, digesta passage rate, and in-vivo digestibility. Performance data was summarized by day and analyzed using a proc-mixed model. The content of physically effective neutral detergent fiber (peNDF) was similar in the HL and SI and lower in the HS, resulting in similar differences among the three corresponding TMRs. In vitro DM digestibility of wheat silage was higher than that of the two hays (65.6% v. 62.8%) resulting in higher in vitro DM digestibility of the SI-TMR compared with the hay-based TMRs (79.3 v. 77.0%). HS-TMR was better than HL- or SI-TMRs at preventing feed sorting by cows after 12 or 24 h eating of the diets. Cows fed HS-TMR consumed more DM and NDF but less peNDF than the other two groups. Average daily rumen pH was similar in the three groups, but daily rumination time was highest in the cows fed HS-TMR. Rumen retention time was longest in cows fed HL-TMR. DM digestibility in cows fed SI-TMR was higher than that of HS and HL groups (65.2%, 61.8% and 62.4%, respectively), but NDF digestibility was similar in the three treatments. The highest intake of digestible DM was observed in cows fed SI-TMR, HS cows were intermediate and HL cows were the lowest. Consequently, cows fed SI-TMR had higher yields of milk, 4% fat corrected milk and energy-corrected milk (47.1, 42.9 and 43.2 kg/day, respectively) than cows fed HS-TMR (45.7, 41.0 and 41.0 kg/day, respectively) or HL-TMR (44.1, 40.3 and 40.3 kg/day, respectively). Net energy production (NEL+M+gain) per kg DM intake was highest in the SI-TMR, lowest in the HS-TMR and intermediate in the HL-TMR (1.52, 1.40 and 1.45, respectively). Animal welfare, as expressed in daily recumbence time and BW gain was similar in the SI and HS groups and higher than the HL cows.

Sacral neuromodulation for the treatment of faecal incontinence following proctectomy.

AIM: This study assessed the effectiveness of sacral neuromodulation (SNM) for faecal incontinence (FI) following proctectomy with colorectal or coloanal anastomosis. METHODS: An Institutional Review Board (IRB)-approved database identified patients treated for FI following proctectomy (SNM-P) for benign or malignant disease, who were matched 1:1 according to preoperative Cleveland Clinic Florida Faecal Incontinence Scores (CCF-FIS) with patients without proctectomy (SNM-NP). Primary outcome was change in CCF-FIS. RESULTS: Twelve patients (seven women) were in the SNM-P group and 12 (all women) were in the SNM-NP group. In the SNM-P group, six patients underwent proctectomy for low rectal cancer and five received neoadjuvant chemoradiation. Five patients had handsewn anastomosis, and one had stapled coloanal anastomosis. One lead explantation occurred after a failed 2-week SNM percutaneous trial. Six patients underwent proctectomy for benign conditions. Within-group analyses revealed significant improvement in CCF-FIS in the SNM-P group (reduction from a score of 18 to a score of 14; P = 0.02), which was more profound for benign disease (reduction from 14.5 to 8.5) than for rectal cancer (reduction from 19.5 to 15). SNM was explanted in 66% and 33% of patients after proctectomy for malignant and benign conditions, respectively. In the SNM-NP group, 41% underwent overlapping sphincteroplasty. One patient received chemoradiation for anal cancer. Within-group analysis for the SNM-NP group showed significant improvement in CCF-FIS (a reduction from 17.5 to 4.0; P = 0.003). There was significant improvement in CCF-FIS in patients without previous proctectomy (mean delta CCF-FIS: 11.1 vs 4.7; P = 0.011). Analysis of covariance (ANCOVA) reaffirmed that controls outperformed proctectomy patients (P = 0.006). CONCLUSION: SNM for FI after proctectomy appears less effective than SNM in patients without proctectomy, with high device explantation rates, particularly after neoadjuvant chemoradiation and proctectomy for low rectal cancer.

Effect of wheat hay particle size and replacement of wheat hay with wheat silage on rumen pH, rumination and digestibility in ruminally cannulated non-lactating cows.

This study examined the effects on intake, diurnal rumen pH changes, rumination and digestibility of feeding ruminally cannulated non-lactating cows in a Latin square design (four cows×four periods) with four total mixed rations (TMRs) typical for lactating cows. TMRs were based on: long wheat hay or short wheat hay, wheat silage or wheat silage+1.5% NaHCO3 buffer, as the sole roughage source (30% of TMR dry matter (DM)). The level of physically effective NDF remaining above the 8 mm screen (peNDF) was similar in the long hay and silage-based TMRs (9.45% to 9.64% of DM) and lower in the short hay TMR (7.47% of DM). The four TMRs were offered individually at 95% of ad libitum intake to avoid orts within 24 h. Cows fed long hay consumed less DM than the short hay and silage groups (9.6 v. 10.5 and 10.8 kg/day, respectively) and sorted against large hay particles at 12 h post-feeding. Under the limitations of this study (non-lactating cows fed at restricted intake) short hay TMR prevented sorting within 12 h post-feeding, encouraged rumination per kg peNDF ingested, and had higher average rumen pH (6.24), whereas preventing sub acute ruminal acidosis (SARA, defined as pH<5.8 for at least 5 h/day). In contrast, the long hay and silage-based groups were under SARA. In vitro methane production of rumen fluid was higher in the hay-fed cows than in their silage-fed counterparts, and in all treatments lower at 1 h pre-feeding than at 6 h post-feeding. In vivo DM and NDF digestibility were similar for the short hay and silage TMRs, and higher than those of the long hay TMR. Under the conditions of this study, addition of 1.5% buffer to the wheat silage TMR had no effect on intake, rumen pH, creation of SARA and digestibility.

Percutaneous cholecystostomy is safe and effective option for acute calculous cholecystitis in select group of high-risk patients.

PURPOSE: In high-risk patients with acute calculous cholecystitis (ACC), percutaneous cholecystostomy (PC) can serve as a bridging option to cholecystectomy [laparoscopic cholecystectomy (LC)] or as definitive treatment. The purpose of this study was to identify predictors of the need for permanent PC. METHODS: Data from 257 PCs performed for ACC (mean age 67.3 ± 14) was collected for a 10-year period. Demographic and clinical characteristics at initial admission, co-morbidities were analyzed. Patients who underwent interval LC were defined as the surgery group (SG; n = 163, 63.4 %) and the remaining patients as the non-SG (NSG; n = 94, 36.6 %). RESULTS: Patients in the SG were significantly younger and had a shorter length of hospital stay (p < 0.01). The rate of coronary artery disease (CAD; 63.2 vs. 20.2 %), chronic renal failure (14.9 vs. 6.1 %), and the mean number of co-morbidities (2.2 vs. 1.4) were significantly higher in the NSG. Sepsis at admission was more common in the NSG (19.1 vs. 4.9 %, p < 0.001). 56 patients (34.4 %) in the SG and 24 patients (25.5 %) in the NSG developed tube-related complications. In hospital mortality was similar between the groups. Multivariate analysis showed that age ≥75, increased alkaline phosphatase (ALK-P), history of CAD, were predictors of PC as a definite treatment in this high-risk group of patients with ACC. CONCLUSIONS: High operative risk due to older age and CAD preclude LC in more than one-third of patients following PC especially presenting with sepsis and elevated ALK-P. This study suggests that PC could be a safe treatment option in this select group of high-risk patients.

The animal gut as a melting pot for horizontal gene transfer.

In this minireview, we examine horizontal gene transfer (HGT) events in the mammalian gastrointestinal tract and their role in the evolutionary adaptation of microorganisms to the gut environment. We explore the notion of the mammalian gut as a melting pot of genetic exchange, resulting in the large extent of HGT occurrence.

Effects of including NaOH-treated corn straw as a substitute for wheat hay in the ration of lactating cows on performance, digestibility, and rumen microbial profile.

This study measured the effects of including 5% NaOH-treated corn straw (T-CS) as a substitute for 15% wheat hay in the control total mixed ration (TMR) of lactating cows on performance, digestibility, and rumen microbial profile. Two groups of 21 cows each, similar in initial performance, were fed individually 1 of the 2 TMR examined. Voluntary dry matter intake of cows fed the control TMR was 4.3% higher than that of the T-CS cows, but in vivo dry matter and organic matter digestibilities of both groups were similar. Crude protein digestibility was higher in the control cows but digestibility of neutral detergent fiber polysaccharides (cellulose and hemicelluloses) was higher in the T-CS TMR. This was followed by 4.6% reduction in rumination time of the T-CS group. A slightly higher milk yield was observed in the control cows compared with the T-CS group; however, milk fat and milk protein content were higher in cows fed the T-CS TMR. This was reflected in 1.3% increase in energy-corrected milk yield and 5.34% increase in production efficiency (energy-corrected milk yield/intake) of the T-CS cows compared with the control. Welfare of the cows, as assessed by length of daily recumbence time, was improved by feeding the T-CS TMR relative to the control group. As a whole, the rumen bacterial community was significantly modulated in the T-CS group in the experimental period compared with the preexperimental period, whereas the bacterial community of the control group remained unchanged during this period. Out of the 8 bacterial species that were quantified using real-time PCR, a notable decrease in cellulolytic bacteria was observed in the T-CS group, as well as an increase in lactic acid-utilizing bacteria. These results illustrate the effect of T-CS on the composition of rumen microbiota, which may play a role in improving the performance of the lactating cow.

Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population.

This study characterizes the effects of concentrated pomegranate-peel extract (CPE) addition to the TMR at levels of 1, 2, or 4% on voluntary intake, in vivo digestibility, milk yield and composition, and profile of rumen bacterial and archaeal populations in lactating Holstein cows. Supplementation of CPE significantly affected the abundance of methanogenic archaea and specific ruminal bacterial species related to cellulolytic activities and soluble sugar and lactic acid fermentation, as revealed by real-time PCR quantification. Furthermore, CPE supplementation had a significant dose-dependent effect on the whole ruminal bacterial community, as determined by automated ribosomal intergenic spacer analysis. These changes were accompanied by a significant increase in digestibility of dry matter, crude protein, and neutral detergent fiber, as well as milk and energy-corrected milk yields in cows fed the 4% CPE supplement. These results suggest that CPE supplementation significantly affects the rumen bacterial communities, which in turn may be related to a beneficial effect on dairy cow performance.

Serum bone alkaline phosphatase and calcaneus bone density predict fractures: a prospective study.

The aim of this study was to assess the ability of serum bone-specific alkaline phosphatase (bone ALP), creatinine-corrected urinary collagen crosslinks (CTx) and calcaneus bone mineral density (BMD) to identify postmenopausal women who have an increased risk of osteoporotic fractures. Calcaneus BMD and biochemical markers of bone turnover (serum bone ALP and urinary CTx) were measured in 512 community-dwelling postmenopausal women (mean age at baseline 69 years) participating in the Hawaii Osteoporosis Study. New spine and nonspine fractures subsequent to the BMD and biochemical bone markers measurements were recorded over an average of 2.7 years. Lateral spinal radiographs were used to identify spine fractures. Nonspine fractures were identified by self-report at the time of each examination. During the 2.7-year follow-up, at least one osteoporotic fracture occurred in 55 (10.7%) of the 512 women. Mean baseline serum bone ALP and urinary CTx were significantly higher among women who experienced an osteoporotic fracture compared with those women who did not fracture. In separate age-adjusted logistic regression models, serum bone ALP, urinary CTx and calcaneus BMD were each significantly associated with new fractures (odds ratios of 1.53, 1.54 and 1.61 per SD, respectively). Multiple variable logistic regression analysis identified BMD and serum bone ALP as significant predictors of fracture (p = 0.002 and 0.017, respectively). The results from this investigation indicate that increased bone turnover is significantly associated with an increased risk of osteoporotic fracture in postmenopausal women. This association is similar in magnitude and independent of that observed for BMD.

Use of bone alkaline phosphatase to monitor alendronate therapy in individual postmenopausal osteoporotic women.

BACKGROUND: Biochemical bone markers are sensitive to the changes in bone turnover that result from treatment of postmenopausal osteoporotic women with antiresorptive therapies. Although information is available on the use of bone markers in monitoring therapy in groups of subjects, less is known regarding how these markers perform in individual patients. METHODS: Serum bone alkaline phosphatase (bone ALP) concentrations, measured with the Tandem(R) Ostase(R) assay, were used to monitor the biochemical response of bone in postmenopausal women with osteoporosis receiving either 10 mg/day alendronate therapy (n = 74) or calcium supplementation (n = 148) for 24 months. RESULTS: Bone ALP decreased significantly from baseline at 3 months (P

Monitoring antiosteoporotic treatment of postmenopausal women using biochemical markers of bone turnover.

Postmenopausal osteoporosis is preventable. An increasing number of antiresorptive therapies including estrogen, selective estrogen receptor modulators (the so-called designer estrogens), bisphosphonates and calcitonins are now available as treatment options for osteoporosis. These agents reduce the level of bone turnover and consequently slow or arrest bone loss and decrease the risk of fracture. However, these therapies are only effective if they are taken as prescribed. Unfortunately, most women who initiate antiresorptive therapy are unwilling or unable to make a long-term commitment to maintain such therapy. The test for biochemical markers of bone turnover can be used to confirm a biochemical response of bone within 3-6 months of initiating therapy, far sooner than the 2 years required for bone density testing. Such information incorporates a timely assessment of the woman's physiologic response with therapeutic compliance. A test result indicating the expected response to treatment may motivate some patients to remain compliant and maintain the therapy for an extended period of time. Bone turnover markers with demonstrated efficacy in monitoring the reduction of bone turnover induced by antiresorptive therapies include bone-specific alkaline phosphatase and osteocalcin (bone formation markers), and free deoxypyridinoline, N-telopeptide and C-telopeptide (bone resorption markers).

Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase.

The performance characteristics of the Tandem-MP Ostase assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3-6.1% with a detection limit of 0.6 microg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 microg/L, S(y/x) = 4.0 microg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 microg/L (heat inactivation) to 6 microg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 microg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1-16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.

Characteristics of a two-site immunoradiometric assay for human skeletal alkaline phosphatase in serum.

This two-site IRMA includes specific monoclonal antibodies for measuring skeletal alkaline phosphatase (B-ALP) in human serum. Assay calibration is based on mass units (micrograms per liter) and was established with purified B-ALP from a human osteosarcoma cell line, SAOS-2. Precision studies demonstrated intra- and interassay CVs of 3-5% and 5-7%, respectively. Relative reactivity studies showed that the assay has a sevenfold preference for detecting B-ALP compared with the liver isoenzyme in serum. The normal reference interval for 478 healthy adults was 5-22 micrograms/L. Method comparison studies showed good correlation between this B-ALP assay (y) and commercially available electrophoretic methods (x) (y = 0.3540x + 20.5, R2 = 0.929) in a pagetic population. Temporal profiles for total ALP, this IRMA B-ALP assay, and B-ALP by electrophoresis in three pagetic patients were parallel. We conclude that this assay demonstrates good analytical performance and would be useful for the clinical assessment of metabolic bone disorders.

Kinetics of oxidation and reduction of high-potential iron-sulfur proteins with nonphysiological reactants.

We have investigated the kinetic and equilibrium oxidation-reduction properties of HIPIP's from four sources: Chromatium vinosum, Thiocapsa pfennigii, Rhodopseudomonas gelatinosa, and a paracoccus sp. These studies focused on the interaction of the various HIPIP's and the iron hexacyanides as a function of ionic strength, pH, and temperature. On the basis of the kinetic and equilibrium data obtained and the known structrual properties of the various HIPIP's a number of conclusions concerning the mechanism of electron transfer by HIPIP can be inferred. (1) The oxidation and reduction of HIPIP by the iron hexacyanides show no kinetic complexity due to heterogeneity or changes in rate-limiting step. (2) The kinetic and equilibrium studies (oxidation-reduction potential) are in good agreement, although a rapid binding equilibrium between HIPIP and the iron hexacyanides is possible. (3) The kinetics of oxidation and reduction of the various HIPIP's studied do not correlate with their oxidation-reduction potentials. (4) The electrostatic interactions between a particular HIPIP and the iron hexacyanides appear to be influenced by the charge on the iron-sulfur cluster with specific amino acid side chains playing a significant but limited role in the interactions leading to electron transfer. (5) The iron-sulfur cluster charge is apparently distributed on the surface of the HIPIP molecule through a network of hydrogen bonds. (6) On the basis of an analysis of the known primary and tertiary structure of the HIPIP's studied, it is concluded that peptide backbone carbonyls and amino acid side chains for the amino acid sequence positions 46-49, 63-65, 78-82, 31-34, and 16-17 define the interaction site of HIPIP and the iron hexacyanides irrespective of the HIPIP source. This is a structural region at which the iron-sulfur cluster is near to the protein surface and in this respect resembles the active site in mitochondrial cytochrome c.