Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization.Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent  Although several experimental treatments intended to detain the progression of CKD have been proposed in the past few decades, only a small minority could be translated into clinical practice. One possible reason for this is that, in general, treatment is initiated in concomitance with the onset of the disease or a few days thereafter, artificially increasing the effectiveness of therapy, since in this manner the pathogenic factors involved are more easily neutralized. Far less encouraging results are obtained if treatment is initiated at later stages, when the much more complex interaction between these factors would require more vigorous therapy and the association of two or more drugs Five-sixths renal ablation (Nx), a widely employed model of chronic kidney disease (CKD), is characterized by severe glomerular and interstitial injury, accompanied by marked hypertension and renal functional loss. Both hemodynamic and inflammatory phenomena are thought to participate in the pathogenesis of renal injury in the Nx model We showed previously In the present study, we investigated whether the L+H association would still arrest the progression of renal injury at more advanced stages of CKD in the Nx model, when extensive renal fibrosis is already present, thereby mimicking more closely the situation prevailing in advanced human CKD. In addition, we sought to determine whether control of systemic hypertension would play a central role in a possible renoprotective effect of the combined L+H treatment in this setting.Two hundred thirty-two adult male Munich-Wistar rats, weighing between 220 and 260 g were utilized in this study. All rats were obtained from a local facility at the Faculty of Medicine, University of São Paulo. All experimental procedures were specifically approved by the local Research Ethics Committee , and developed in strict conformity with our institutional guidelines and with international standards for manipulation and care of laboratory animals. All rats were monitored daily for body weight and general condition. Rats that were in bad condition, presumably due to end-stage renal failure were euthanized by an overdose of anesthetic. Five-sixths renal ablation (Nx) was performed in a single-step procedure after ventral laparotomy under anesthesia with ketamine 50 mg/kg and xylazine 10 mg/kg im. The right kidney was removed and two or three branches of the left renal artery were ligated, resulting in the infarction of two-thirds of the left kidney. Sham-operated rats underwent anesthesia and manipulation of the renal pedicles, without any removal of renal mass. After surgery, all animals received enrofloxacin and, after full recovery, were given free access to tap water, fed regular rodent chow containing 0.5 Na and 22% protein , and kept at 23±1°C and 60±5% relative air humidity, under an artificial 12–12 hour light/dark cycle.The possibility of regression or prevention of renal injury was analyzed in 2 protocols representing a moderately advanced (Protocol 1) and a very advanced (Protocol 2) stage of chronic nephropathy.albV) were determined in all Nx rats. Those failing to increase TCP above 170 mmHg or UalbV above 40 mg/day were not included in the study. Fifteen Nx rats were killed at this time and were used as pretreatment control subjects (group Nxpre). The remaining 74 Nx rats were divided into 4 experimental groups: Nx+V (n = 22); Nx+L (n = 15), Nx receiving losartan potassium (L), 50 mg/kg diluted in the drinking water; Nx+LH (n = 17), receiving L 50 mg/kg plus hydrochlorothiazide (H) 6 mg/kg diluted in the drinking water; Nx+AHHz (n = 20), receiving amlodipine besylate 5 mg/kg plus H 6 mg/kg plus hydralazine chloride (Hz) 12 mg/kg. This last group was included to evaluate the degree of renal protection that would be afforded by decreasing TCP to a similar extent as in group LH, without suppressing the renin-angiotensin system. Nx rats were distributed in such a way that initial body weight (BW), TCP and UalbV were similar among experimental groups. All treatments were maintained for 90 days. A group of 15 Sham-operated rats receiving no treatment was followed for the same time.Sixty days after Nx, tail-cuff pressure (TCP) and daily urinary albumin excretion . The remaining 85 Nx rats were distributed in the same way as for Protocol 1, among groups: Nx+V (n = 26); Nx+L (n = 19); Nx+LH (n = 20); and Nx+AHHz (n = 20), treatments being maintained for 90 days. A group of 23 untreated sham-operated rats was followed for the same time.One hundred twenty days after Nx, TCP and UalbV was evaluated by radial immunodiffusion and TCP was measured using an optoelectronic automated device  under light restraining and after light warming. To avoid any interference of stress, all rats were preconditioned to the procedure, and were invariably calm at the time of TCP determination. In addition, TCP was taken as the average of at least three consecutive measurements that varied by no more than 2 mmHg, to ensure that BP was stable before readings. At the end of the study, blood glucose (BG) and plasma triglycerides (Tg) were measured in blood taken from a tail vein after 12-hour fasting. On the following day, rats were anesthetized with ketamine, 50 mg/kg and xylazine 10 mg/kg im, and blood was collected from the abdominal aorta for measurement of serum creatinine (SCr), aldosterone (ALDO) and potassium (K+) concentration. The kidneys were then retrogradely perfusion-fixed through the abdominal aorta with Dubosq-Brazil solution after a brief washout with saline to remove blood from the renal vessels. After weighing, two midcoronal renal slices were postfixed in buffered 4% formaldehyde and embedded in paraffin using conventional sequential techniques. Histomorphometric and immunohistochemical analyses of the renal tissue were performed in 4-µm-thick sections.Monthly UTg and BG were measured in blood samples obtained from a tail vein Tg was measured using a commercially available enzymatic kit . BG concentration was determined using a reﬂectometric method .Cr was determined using a commercially available kit . A radioimmunoassay kit  was used to determine ALDO. Serum K+ was measured using an electrolyte analyzer .SMorphometric evaluations were always performed in a blinded manner by a single observer. The extent of glomerular injury was estimated by determining the frequency of glomeruli with sclerotic lesions, as described previously Immunohistochemistry was performed on 4-µm-thick sections, mounted on glass slides precoated with 2% silane. Sections were deparaffinized and rehydrated by conventional techniques, then heated in citrate buffer for antigen retrieval and incubated overnight with the primary antibody at 4°C. For the negative control experiments, incubation with the primary antibody was not performed. The following primary antibodies were used: monoclonal mouse anti-rat ED-1 antibody for macrophage detection ; monoclonal mouse anti-rat proliferating cell nuclear antigen (PCNA) ; monoclonal mouse anti-alpha-smooth muscle actin (α-SMA) ; polyclonal rabbit anti-human AII , polyclonal rabbit Anti-Collagen I  and polyclonal rabbit anti-rat thiazide-sensitive Na-Cl cotransporter, NCC .For ED-1 detection, sections were preincubated with 5% normal rabbit serum to prevent nonspecific binding, then incubated overnight at 4°C with the primary antibody diluted in bovine serum albumin (BSA) at 1%. After rinsing with Tris-buffered saline (TBS), sections were incubated with an appropriate secondary antibody, then with an alkaline phosphatase anti-alkaline phosphatase (APAAP) complex . Sections were developed with a fast-red dye solution . For AII and α-SMA detection a streptavidin-biotin complex for alkaline phosphatase  was used. Nonspecific binding was prevented with normal horse serum diluted at 1∶20 in nonfat milk at 2% in TBS. Primary antibodies for AII and α-SMA were diluted in BSA at 1∶400 and 1∶800, respectively. Sections were developed in the same manner as for ED-1 detection. For assessment of PCNA-positive cells and the percentage of the renal area occupied by collagen I, sections were pretreated with 30% hydrogen peroxide in methanol and preincubated with normal horse serum as described. The primary antibodies were diluted at 1∶100 (PCNA) and 1∶200 (collagen I), in nonfat milk at 2% in TBS. The EnVision Labelled Polymer for peroxydase  was used before development with DAB substrate .Double immunostaining was used to visualize the proliferation activity of distal convoluted tubules (DCT). Identification of DCT was performed by detection of NCC. Sections were pretreated with 30% hydrogen peroxide in methanol and preincubated with avidin and biotin blocking solutions . Nonspecific staining was then prevented with normal goat serum diluted at 5% in BSA at 1% in TBS. Sections were incubated overnight with primary antibody against NCC diluted at 1% in BSA at 1% in TBS. Appropriate biotinylated secondary antibody was applied and Streptavidin-AP solution  was used, followed by development with fast-red dye solution . Sections were then preincubated once again with avidin and biotin blocking solutions, followed by prevention of nonspecific staining with a mixture of normal horse and rabbit sera diluted at 2 and 5%, respectively, in 2% nonfat milk in TBS. Sections were then incubated overnight with the primary antibody against PCNA, 0.01% in a solution containing 1% BSA and 2% nonfat milk diluted in TBS. Appropriate biotinylated secondary antibodies were applied and the LSAB-HRP kit  was used for PCNA detection. Sections were developed with DAB substrate . All sections were counterstained with Mayer’s hematoxylin, dehydrated and covered with Permount Mounting Media .2) were examined. The percentage of cortical interstitial area occupied by α-SMA was estimated by the same point-counting technique employed to evaluate %INT, excluding positively stained blood vessels, while interstitial area occupied by collagen I was measured with an image processing software .The renal density of macrophages, proliferating cells and AII positive cells was evaluated in a blinded manner at 200× magnification. For each section, 50 microscopic fields .Differences among different groups were analyzed using one-way analysis of variance (ANOVA) with pairwise post-test comparisons by the Neuman-Keuls method Survival data for Protocol 1 are shown in Body weights observed in Protocol 1 are shown in albV with time in Protocol 1 is shown in albV, reaching values tenfold higher than in S at 150 days (p<0.05 vs. S). In Group Nx+L, UalbV remained markedly elevated along the study, although final values were nearly 30% lower than in Group Nx+V (p<0.05). By contrast, combined LH treatment promoted UalbV regression, keeping it close to S values until the end of the study (p>0.05), although it should be noted that, since the nephron number had been reduced to 1/6th of normal, albuminuria per nephron was still higher in this group compared to S. Treatment with AHHz was unable to lower UalbV, which was always similar to that seen in Group Nx+V. The evolution of UalbV in Protocol 2 is represented in albV was always elevated in Group Nx+V, being lowered by a small but significant amount by L treatment. Triple AHHz therapy had little effect on UalbV. Even at this advanced stage, the LH treatment reduced UalbV to levels that approached those observed in S, remaining at these low levels until the end of the observation period (p>0.05 vs. S).The variation of UCr) for Protocol 1 are shown in pre) SCr was twice as high as in Group S (p<0.05). At the end of Protocol 1, 150 days after renal ablation, Scr was increased further (p<0.05 vs. S and Nxpre), indicating progression of the nephropathy. Treatment with L attenuated the loss of renal function, maintaining Scr at levels comparable to those observed in the pretreatment group (p>0.05 vs Nxpre and p<0.05 vs. Nx+V). Similar functional protection was observed with the LH association (p>0.05 vs Nx+L). The AHHz scheme had no effect on SCr, which increased over time and was indistinguishable from the Nx+V value at the end of the study. In Protocol 2 . Animals in Group Nx+V showed severe loss of renal function 210 days after renal ablation, as indicated by a marked increase in SCr (p<0.05 vs. Nxpre). Treatment with L or AHHz had little effect on SCr, which attained levels similar to those seen in Group Nx+V at the end of the study (p>0.05). By contrast, rats treated with the LH combination exhibited final SCr levels that were significantly lower than in the remaining groups, and similar to those verified in Group Nxpre (p>0.05).Serum creatinine concentrations (Sotocol 2  Scr was + concentrations for Protocol 1 are shown in pre) serum K+ was significantly increased compared to S. Animals in group Nx, at both 60 and 150 days after nephrectomy, showed an increase in serum potassium (p<0.05 vs S). Treatments with L or LH had no effect on serum K+ at the end of the period of observation. The AHHz regimen strongly decreased serum K+, which was similar to that observed in the S group 150 days after renal ablation. Values for Protocol 2 are shown in pre group, 120 days after nephrectomy (p<0.05 vs. S), and in Group Nx+V, examined at the end of the study. Treatment with L aggravated hyperkalemia, serum K+ reaching values significantly higher than those observed in the Nxpre group (p<0.05). In animals receiving LH therapy, serum K+ was ameliorated compared to the Nx+L group (p<0.05), being similar to that seen in the Nxpre group (p>0.05). As observed in Protocol 1, treatment with AHHz significantly reduced serum K+, which was brought to levels similar to those observed in the S group (p>0.05).Serum Kpre . Triple therapy promoted no change in ALDO concentration, which remained comparable to that in Group Nx+V (p>0.05). In Protocol 2, the pretreatment ALDO concentration (Group Nxpre) was significantly higher than in Group S (p<0.05), a difference that persisted in Group Nx+V. Monotherapy with L promoted a significant reduction compared to Group Nx+V (p<0.05). LH treatment drastically reduced circulating ALDO below pretreatment levels . In Group Nx+AHHz serum ALDO was similar to that in Group Nx+V.The values for aldosterone (ALDO) in both protocols are shown in pre). Glomerular injury progressed in untreated animals, %GS exceeding 30% in Group Nx+V 150 days after nephrectomy (p<0.05 vs. Nxpre). Treatment with L alone partially prevented the progression of glomerular lesions, although %GS in Group Nx+L was not significantly different from that seen in untreated rats (p>0.05 vs. Nxpre and Nx+V). Glomerular protection was evident in the group receiving the LH association, in which the value for %GS was nearly identical to that observed in Group Nxpre. In the group treated with the AHHz association, the frequency of glomerular sclerotic lesions was not limited, progressing in a similar fashion as in the untreated group. In Protocol 2, %GS already reached 27% 120 days after renal ablation (Group Nxpre). After 210 days post-ablation, almost 60% of glomeruli exhibited sclerotic lesions (p<0.05 vs. Nxpre). Treatment with L failed to prevent the progression of %GS, which reached values that were lower than those found in untreated rats, but significantly higher than pretreatment values. In rats treated with LH, %GS reached final values that were significantly lower than in Group Nx+V or Nx+L, and similar to those seen in Group Nxpre. In animals treated with AHHz, %GS behaved in a similar manner as in the untreated group.Representative glomeruli seen at the end of Protocol 1  are shown in pre exhibited high %INT values compared to S (p<0.05). Little progression was seen in untreated rats 150 days after ablation (p<0.05 vs. Nxpre). None of the treatments had a significant effect on %INT at this phase. pre in comparison with S. At 210 days post-ablation, %INT was significantly increased in Group Nx+V compared with pretreatment values (p<0.05 vs. Nxpre). L monotherapy was unable to attenuate the progression of interstitial expansion, %INT reaching similar values as in Group Nx+V. By contrast, the LH association completely prevented the progression of %INT, which remained at similar levels as in Group Nxpre. No protection against interstitial expansion was obtained in rats treated with the AHHz association.Representative microphotographs of cortical interstitial area are shown in The presence of collagen I in the renal tissue, detected by immunohistochemistry, is shown in pre). Rats treated with L only or with the AHHz association showed values that were similar to those seen in Group Nx+V (p>0.05), whereas combined LH treatment effectively prevented the increase of interstitial α-SMA with time (p>0.05 vs. Nxpre). Parallel results were observed in Protocol 2, with the only difference that the fractional α-SMA area at the end of the study was significantly smaller in Group Nx+LH than in Group Nxpre.Representative microphotographs of interstitial α-SMA obtained 150 days (Protocol 1) and 210 days (Protocol 2) after renal ablation are shown in pre compared to S (p<0.05). This parameter showed a progressive nature, reaching final values (150 days after ablation) that were significantly higher than those observed in Group Nxpre. L and LH treatments, but not the AHHz association, prevented the intensification of tubulointerstitial macrophage infiltration (p>0.05 vs. Nxpre). Data for Protocol 2 are shown in pre) 120 days after renal ablation (p<0.05 vs. S), with progression to final values (210 days after Nx) that were significantly higher than in the Nxpre group. All treatments prevented progression of macrophage infiltration, although this parameter was numerically lower in Group Nx+LH.pre), exhibiting further elevation 150 days after Nx (p<0.05 vs. Nxpre). Neither L monotherapy nor the AHHz association were able to prevent the increase in the number of AII-positive cells, while the LH combined therapy reduced this parameter to values that were similar to those found in Group Nxpre, and significantly lower than in each of the other Nx groups. Entirely parallel results were obtained in Protocol 2 (Representative microphotographs showing cells staining positively for AII are depicted in otocol 2 .pre). Interstitial, but not tubular proliferation was significantly increased in untreated Nx rats compared to Nxpre. Among treatments, only the LH association was able to prevent, and even reverse, cell proliferation in both tubular and interstitial compartments, bringing these parameters to levels that were similar to those found in S. In Protocol 2, pretreatment values for tubular and interstitial proliferation were higher than in S. No progression of these parameters was seen in untreated Nx rats 210 days after renal ablation. At this time, interstitial proliferation was less intense in rats that received L monotherapy or the AHHz combined therapy than in Group Nx+V, but remained at levels similar to those observed prior to treatments. No difference was observed at this time among Groups Nx+V, Nx+L and Nx+AHHz regarding tubular cell proliferation. In rats treated with the LH association, values for PCNA-positive cells in all three compartments were significantly lower than in the Nx+V group, and even lower than in the pretreatment group, indicating that treatment promoted regression of cell proliferation in this group.Representative microphotographs of renal proliferating cells are shown in pre. L monotherapy and the AHHz regimen promoted no significant change in DCT proliferation. By contrast, the LH association reduced DCT proliferation below pretreatment levels, indicating regression of this parameter. Parallel results were obtained in Protocol 2, with the exception that the differences between Group Nx+LH and either Nx+L and Nx+AHHz were now significant.Representative microphotographs of proliferation in the DCT, obtained through simultaneous staining for PCNA and NCC, are shown in pre compared with S. At the end of the study (150 days after Nx) BG was slightly elevated in all groups, although only in Groups Nx+L and Nx+AHHz was this value significantly different from that in S. In Protocol 2 , BG was significantly increased in untreated Nx+V rats (p<0.05 vs. S). None of the treatments was able to normalize BG, which remained significantly increased compared to S in all groups of treated rats. Serum triglyceride concentrations (Tg) for Protocol 1 are given in pre), and remained elevated at the end of the study. Only with the LH therapy did Tg fall to values significantly lower than in the Nx+V group, and similar to pretreatment values.In Protocol 1 , BG leveotocol 2 , BG was As described earlier In most previous studies of the Nx model, treatments intended to prevent or ameliorate CKD were initiated immediately after nephrectomy or at most a few weeks after the procedure. At this initial phase, structural injury in the remaining renal tissue is incipient or only mild, and can be more easily prevented. We showed previously In the present study, we compared the response of Nx rats to L monotherapy and the LH association, started at two different times after nephrectomy –60 and 120 days. Whereas the first time point simulated a condition of low nephron number with relatively mild renal inflammation, the second one –120 days after nephrectomy, never used before as a starting point for therapy – represented a situation in which drastic nephron reduction leads to high mortality and coexists with severe ongoing renal injury, with extensive glomerulosclerosis, interstitial inflammation and tubular atrophy, thus mimicking more closely the conditions prevailing in patients with advanced CKD, and their response to therapy.Monotherapy with L provided only partial protection against mortality and the progression of renal injury/inflammation, especially when started 120 days after ablation. These results are consistent with previous observations albV and hypertension to levels below those observed before treatment, arresting the progression of renal injury and the decline of renal function, and dramatically reducing mortality. The factors that might explain this remarkable synergistic action are unclear. One obvious possibility is the striking BP reduction obtained with LH treatment In both protocols, the effectiveness of treatment with L was sharply increased when associated with H, promoting regression of UInsulin resistance and dyslipidemia may accompany CKD, aggravating renal injury Although thiazides have been used for decades in the treatment of hypertension, and although their value as antihypertensive agents in CKD has recently been affirmed In summary, the present results suggest that the L+H association arrests the progression of renal injury in the Nx model, even when initiated at advanced stages of the nephropathy, with ongoing GS and interstitial inflammation and fibrosis. Our results do not support the concept that thiazides lose their effect in advanced CKD, nor do they reproduce the metabolic effects attributed to chronic thiazide use. The renoprotective action of the LH association cannot be explained by its antihypertensive effect. Mechanisms possibly involved in this protective action are a fall in intraglomerular pressure, the sharp decrease observed in the filtration of albumin and in the production of aldosterone, as well as an antiproliferative effect. Additional studies are needed to investigate whether these renoprotective effects can also be obtained in human CKD even when treatment is initiated at very advanced stages.


Studies have shown inconsistent results regarding the association between dietary factors across the lifespan and breast density and breast cancer in women. Breast density is a strong risk factor for breast cancer, and the mechanism through which it influences cancer risk remains unclear. Breast density has been shown to be modifiable, potentially through dietary modifications. The goal of this paper is to summarize the current studies on diet and diet-related factors across all ages, determine which dietary factors show the strongest association with breast density, the most critical age of exposure, and identify future directions. We identified 28 studies, many of which are cross-sectional, and found that the strongest associations are among vitamin D, calcium, dietary fat, and alcohol in premenopausal women. Longitudinal studies with repeated dietary measures as well as the examination of overall diet over time are needed to confirm these findings. Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death among women . Alcohol A literature search of the PubMed database of the United States National Library of Medicine was conducted to find human studies that evaluated the associations between BD measures and diet isn the form of either single nutrients or whole dietary patterns. Both observational and diet intervention studies conducted at any stage of the lifespan were considered. Observational studies were included if they had recorded individual's dietary intake of foods or energy with dietary assessment tools such as a dietary recall (DR), food frequency questionnaire (FFQ), food record (FR), or other relevant assessment tools. Relevant studies were identified using the following search terms in multiple combinations: “adolescent diet and breast density,” “diet and breast density,” “childhood and breast density,” “diet and parenchymal patterns” and “mammographic breast density and diet.” The search was limited to full-text publications written in English. As illustrated in r = 0.73) [r = 0.26) [BD can be measured two (2D) and three dimensionally (3D), with the most common being through 2D mammography. Mammography measures the area of dense tissue (ADT) and the total area of the breast. Percent dense area (PDA) is often reported and is estimated as the proportion of dense fibroglandular tissue area to total breast area . Area of = 0.73) , but thi = 0.26) . In addi = 0.26) –41. Thes = 0.26) .In general, PDA is higher in premenopausal women compared to postmenopausal women as well as in postmenopausal women who use hormone replacement therapy (HRT), and in both pre- and postmenopausal women with a lower BMI; and is lower in women who are parous, experience their first birth at a younger age, or are smokers , 43, 44.This review will focus primarily on diet and diet-related factors and their potential effects on both PDA and ADT with only limited attention to endogenous risk factors that are well-studied and not modifiable. A summary of these findings can be found in Tables Much of breast development occurs during puberty; thus, factors such as childhood diet that influence the timing of puberty could potentially affect BD , 47. Thr2, 87.7 cm2 and 53.1 cm2 in unexposed, moderately, and severely exposed, respectively (Ptrend = 0.03). Although not significant, the women who were severely energy restricted at this age also had a larger ADT and higher PDA. However, because only 15 subjects were severely restricted, results should be interpreted cautiously. Haars and colleagues  examinedThe three studies that examined childhood diet and its effect on adult BD measures did not find associations with PDA or ADT although, in the study of the Dutch famine, severe caloric restriction early in life was significantly inversely associated with ANDT later in life . In thisMost of breast development occurs during puberty, and diet during this time could have long-term effects on BD in adulthood. One of five studies we found a significant association between diet during adolescence and BD in adulthood . In the The remaining four studies, including 3 observational studies and one clinical trial, found no associations between dietary components or alcohol consumption during adolescence and BD in adulthood , 17, 18.The majority of studies that have evaluated associations of diet with BD assessed the effects of adult diet. A total of 26 epidemiological studies and randomized controlled trials that examined dietary intake and BD among adult women are included in this paper.  Three studies examined the association of total energy intake in adulthood with BD measures . In a naPtrend = 0.04). In a sample of 31 BC patients, women in the highest quartile of total fat (mean % energy (E) = 42.04) compared to the lowest quartile of intake (mean %E = 34.72) were significantly more likely to be classified as a P2 + DY (high density) pattern compared to the N1 + D1 (low density) pattern (P < 0.01) [Eight studies –32, 51 h < 0.01) . QureshiP trend = 0.009) [P trend = 0.02) [E of 19.27 from SFA in the highest quartile were more likely to be classified as having a high-risk PDA compared to those who consumed a mean %E of 15.42 from SFA in the lowest quartile (P ≤ 0.05). In contrast, a significant inverse association was observed with SFA in a subset of 283 premenopausal women from the MBCFSC; mean PDA was 37% in those with the highest SFA intake compared to 44% in the lowest consumers after controlling for relevant confounders (P trend = 0.03) [Individual fatty acids have also been examined with saturated fatty acids (SFAs) generally being positively associated with increased BD measures. In an analysis based on 645 pre- and postmenopausal women ages of 40–62 years enrolled in the Canadian National Breast Screening Study (CNBSS), SFA intake was significantly positively associated with PDA. Mean PDA was 44.2% in the highest quartile of SFA intake compared to 38.6% in the lowest (= 0.009) ; however = 0.02) . Qureshi = 0.02)  also sho = 0.02)  observed = 0.03) . No assoE = 5.65 versus 4.70) and n-6 fatty acids (Mean %E = 4.69 versus 3.81) was also significantly associated with being classified as a P2 or DY (high density) versus an N1 or P1 (low density) Wolfe parenchymal pattern (P < 0.05). Vachon et al. [P = 0.05). Similar results were observed with the PUFA : SFA ratio in this study.  The essential PUFA, linolenic acid, was inversely associated with PDA in a mediterranean population of both pre- and postmenopausal women. Women in the highest tertile of intake had 31% lower odds of being classified as high PDA . Elevaten et al.  examinedE = 14.22) were more likely to have a high PDA compared to those in the lowest quartile . Far fewer associations between dietary factors and BD measures were observed in postmenopausal women, with only increased consumption of MUFAs being significantly associated with high PDA even though the difference in MUFAs as percent energy between the high and low density groups was small . A small number of randomized controlled trials (RCTs) have also been conducted to examine dietary fat and BD and have yielded mixed results [ Finally, Nordevang and colleagues  found th results –37. ThesPtrend = 0.08). When the type of alcohol was examined, comparable results were observed with white wine in postmenopausal women only; however, an inverse association was observed with red wine in postmenopausal women with “nondrinkers” having a mean PDA of 34% compared to 32% for those consuming ≤1 serving/wk and 28% for those consuming ≥2–4 svg/wk (Ptrend = 0.02) [In their 2001 review, Singletary and Gapstur  conclude = 0.02) . The aut = 0.02) . Tseng e = 0.02)  and Sala = 0.02)  also looMaskarinec et al.  conducteP < 0.01). Similar results were found when intake from food and supplements were analyzed together. Vitamin D and calcium have been linked to cellular growth and differentiation in breast tissue , 57 and Out of the remaining seven studies, two included only postmenopausal women and neither found an association between vitamin D and calcium intake and BD , 20. An P = 0.003), which was consistent with the rise and fall in serum vitamin D across the seasons. Few studies account for season in which BD was assessed. However, it may be important to consider endogenous vitamin D synthesis in response to sunlight in addition to that contributed by food sources. The biologically active form of vitamin D may decrease BD via its antiproliferative properties or tissue-specific effects due to breast tissue possessing 1-α-hydroxylase, which converts inactive 25(OH)D to active 1,25(OH)2D [2D helps to regulate cell growth and promote terminal differentiation which promotes cellular resistance from carcinogenic factors [Overall, this research suggests that vitamin D and calcium are inversely associated with BD in premenopausal women. It is critical to note that as calcium and vitamin D increased from <500 mg/d and <100 IU/d to >1,750 mg/d and >700 IU/d, respectively, PDA decreased in a dose-response fashion with clinically relevant decreases in PDA between 8 and 12% among premenopausal women , 23. Thi25(OH)2D . The loc factors . Premeno factors , 62. VitTen studies have evaluated intakes of carbohydrates, protein, and many other nutrients and their association with BD measures. Eight studies , 34, 51 Although few significant associations are observed among postmenopausal women; both Nagata et al.  and Salaβ-carotene, vitamin C, and potassium, whereas women in the highest tertile of tomato sauce intake had 34% higher odds of having a high PDA [In a study evaluating dietary factors and mammographic patterns in a Mediterranean population, both pre- and postmenopausal women in the highest tertiles of the following foods and nutrients had 27–34% lower odds of having a high PDA: total vegetables, cheese, high PDA . Similarhigh PDA . Consisthigh PDA . Among phigh PDA . Comparahigh PDA . Ptrend = 0.009). No association was observed in postmenopausal women. Vachon et al. [Only one study to date has examined multivitamin/multimineral (MVMM) supplement intake and BD outcomes. Bérubé and colleagues  found thn et al.  also foun et al. . In conclusion, the foods or nutrients that were shown to be inversely associated with BD may be, in part, tied to IGF/IGFBP levels and oxidative stress reduction. BD has been associated with increased levels of oxidative stress as evidenced by malonoyldialdhyde (MDA) excretion  and IGF/a posteriori dietary patterns and their association with BD and one study examined the influence of Mediterranean Diet ) on BD measures. Dietary patterns were analyzed cross-sectionally in a British cohort and the MBCFSC [P = 0.02) [ Analysis of dietary patterns has recently gained popularity in dietary assessment research, as they capture total diet and are more stable over time than the consumption of single nutrients or foods . Two stue MBCFSC , 33. Afte MBCFSC . In the e MBCFSC . When al = 0.02) . These pP = 0.0002) among current smokers but not among nonsmokers after controlling for relevant confounders including menopausal status [Tseng et al.  cross-sel status . Vegetabl status .Overall, it appears that dietary patterns high in antioxidant-containing foods are inversely associated with BD in smokers, who may be experiencing a higher level of oxidative stress than nonsmokers. Other research has shown a positive association between BD and MDA, which is a marker for lipid peroxidation and oxidative stress . P = 0.01) [The epidemiological evidence described above suggests that diet is associated with BD measures and that BD has the potential to be modified. As a result, researchers have conducted clinical trials to examine the association between specific dietary factors with BD outcomes . Boyd et = 0.01) . The eff = 0.01) . After s = 0.01) . Martin et al.  completeAs previously described in the vitamin D and calcium section, a one-year calcium and vitamin D supplementation trial was conducted through the WHI to examine the effects on mammographic PDA in postmenopausal women . Despite Data from observational studies suggest that the strongest associations between diet and BD measures are among vitamin D, calcium, dietary fat, and alcohol and are found in adult premenopausal women. However, the few clinical trials that have evaluated these associations have failed to demonstrate a significant change in breast density with various dietary interventions. This could be because the foods/nutrients evaluated truly do not influence breast density or could be due to aspects of the study design including duration of the intervention, dose, sample size, or inclusion of predominantly older women in whom breast tissue may be less susceptible to dietary influences. This paper has critically examined 28 studies and has identified strengths and weaknesses as well as highlighting several potential directions for new research to advance the field. Many of these studies are cross-sectional in nature and often focus just on PDA. In addition to this, the majority of women who receive mammograms overall and in these studies are >40 y; an association between dietary factors and BD measures could be undetected if the critical dietary exposure occurred much earlier in life (and was not measured) before breast tissue is fully differentiated and potentially more vulnerable to exogenous influences.The majority of studies included in the paper assessed BD using 2D mammography. Even though estimates of BD obtained by mammography and 3D modalities such as MRI are highly correlated in the general population and in women with less dense breasts , correla Many studies examined the association of diet with PDA but not the ADT. Fewer associations are observed with the ADT compared to PDA; however, results should be reported when available in order to be more comprehensive, improve comparisons across studies, and enhance interpretability in relation to potential physiological mechanisms. Very few studies controlled for the phase of the menstrual cycle at the time of mammography. Because data on variation of breast density over the menstrual cycle are conflicting –71, it sTo date, most studies of the association of diet with BD have been cross-sectional. Longitudinal studies that measure diet and BD over the life course are needed. Studies that evaluate the influence of diet during adolescence, when most breast development occurs, on adult BD could be particularly enlightening. Support for an association of diet with BD from observational studies is stronger for premenopausal women. However, a limited number of short-term clinical trials do not show conclusive evidence that dietary factors influence BD. Clinical trials in younger women could be informative and may provide more definitive results. Lastly, more research on dietary patterns as they relate to BD are needed.


O-deethylase (EROD) activity. This study was aimed at developing this assay in the Cape hake species Merluccius capensis and Merluccius paradoxus (considered one stock). Microsomal fractions were obtained from frozen fish liver samples by differential centrifugation. Fluorimetric and spectrophotometric analysis of the EROD assay resulted in the spectrophotometric (at 572 nm) detection method being selected, as this method resulted in a lower degree of variability and demonstrated higher reproducibility. The activity in the EROD assay was enhanced in the presence of NADPH, and the addition of dicumarol (phase II enzyme inhibitor) to the reaction mixtures prevented the underestimation of this assay by the inhibition of DT-diaphorase. In summary, an EROD assay was established for use in Cape hake species.Cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds, for example, polychlorinated biphenyls (PCBs) and the induction of this protein is often measured in terms of CYP1A-mediated 7-ethoxyresorufin- In recent years the increased production and release of organic trace pollutants, for example, herbicides, metals, polychlorinated biphenyls, alkylphenols, insecticides and industrial effluent mixtures into the marine environment have increased concern and awareness into the bioaccumulation, bioconcentration, and biomagnification of these pollutants in marine organisms –3. The pFish models have played a significant role in toxicological studies used to assess the state of aquatic environments , 12. Thep-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) [The aim of the present study was to optimize the cytochrome P450 monooxygenase 1A (CYP1A) 7-ethoxyresorufin deethylation (EROD) reaction for Cape hake. The superfamily of cytochrome P450s are heme containing proteins which regulate the metabolism (phase I metabolism) of several xenobiotic and endogenous compounds . CYP1A bs (PCBs) –19. CYP1s (PCBs) . CYP1A is (PCBs) –22. The induction of CYP1A is most commonly measured in terms of EROD activity, as this indirect strategy has proven to be cost-effective and sensitive even in a complex mixture of compounds . The EROMerluccius capensis and Merluccius paradoxus (Cape hake) were used in this study as the model species for the development of the EROD assay. Cape hake belong to the family Gadidae and coinhabit Namibian and South African waters [M. capensis and M. paradoxus are morphologically similar and the differences that separate them, for example, differences in the number of vertebrae are very minor [M. capensis occurs predominantly off the coast of Namibia and the south coast of South Africa [M. paradoxus overlaps with the region inhabited by M. capensis but this species is predominantly found along the west coast of South Africa [Merluccius species are extremely limited [n waters . Their gn waters . M. capery minor , 30. In h Africa . The dish Africa . Cape hah Africa . These fh Africa . Cape hah Africa  and stud limited .dl-isocitric acid trisodium salt, dicumarol, dl-dithiothreitol (DTT), bovine serum albumin (BSA), Bradford's reagent, and Ponceau S red and nitrocellulose membrane were supplied by Sigma-Aldrich, South Africa. Ethylenediamine tetraacetic acid disodium salt (EDTA), glycerol, glycine, sodium dodecyl sulphate (SDS), tris(hydroxymethyl)-aminomethane, Tween 20, methanol, magnesium sulphate, acetone, and 2-[4-(hydroxyethyl)-1-piperazinyl-ethanesulfonic acid (HEPES) were supplied by Merck, South Africa. Nicotinamide adenine dinucleotide phosphate (NADPH) was supplied by Calbiochem, South Africa. The BM chemiluminescence western blotting kit (mouse/rabbit) was supplied by Roche, South Africa, and rabbit anti-fish CYP1A peptide was supplied by Biosense Laboratories AS, Norway. Resorufin, 7-ethoxyresorufin, Coomassie Brilliant Blue R250, methanol, M. capensis and M. paradoxus, n = 11). All Cape hake samples were transported at approximately −8 to −10°C and arrived frozen. Samples were thawed at 4°C overnight , and the total length (cm) and weight (g) were measured to determine Fulton's condition factors (CFs). Livers were excised and the weight noted (g). Excised samples were stored at −20°C until further analysis (thawed liver samples were retained for no longer than a month).Frozen Cape hake samples were obtained from Sea and Coasts in Cape Town, South Africa and Balobi Trading, Mossel Bay, Eastern Cape, South Africa /pellet 2 (P2), which was resuspended in 1 : 1 (w/v) resuspension buffer  glycerol), pH 7.4. PMS and MS fractions were retained and stored at −20°C. Protein concentrations for the two fractions were determined according to Bradford [Liver preparations were carried out using a modified protocol of Nilsen . All preBradford  at 595 nBradford . μL), 7-ethoxyresorufin (0.097 mg/mL in methanol), and tris-NaCl (TN) buffer . The reaction mixture was incubated in a Labnet dry bath at 23°C for 15 minutes, after which time the reaction was terminated by the addition of 500 μL ice cold methanol (99.5%). Samples were then centrifuged at 6,000 × g in a Heraeus Megafuge 1.0 R for 20 minutes at 4°C to remove any cellular debris that could interfere with the results. The resultant supernatant was analyzed spectrophotometrically at 572 nm [X spectrophotometer using KC Junior software. Results were also analyzed using a Hitachi Spectrofluorometer F2500, using excitation and emission wavelengths of 510 and 585 nm, respectively [EROD analysis was performed using a modified method described by Pikkarainen , on the t 572 nm , 40 in aectively . All samμL) was spotted onto a nitrocellulose membrane and incubated at 23°C until sample spots had completely dried. Immunoblot reactions (BioRad instructions) were carried out using primary (rabbit anti-fish CYP1A peptide) and secondary (anti-mouse/rabbit-antibody-horseradish peroxidase conjugate (POD)) antibody dilutions of 1 : 5000. The homogenization buffer was used as a negative control. No positive control was included as no commercial fish CYP1A for any fish species was available at the time of this study.Dot blot analysis was used to determine the presence of CYP1A in different microsomal fractions that were obtained from differential centrifugation. A modified dot blot analysis was carried out as described by Desantis et al. . The micμL). The NADPH dependence of the EROD assay was also assessed by running parallel reactions in the absence and presence of the coenzyme (0.1 M). All experiments were conducted in triplicate with appropriate enzyme and substrate controls. Results were analyzed at 572 nm with a Powerwavex spectrophotometer using KC Junior software.EROD assay conditions were optimized for Cape hake with respect to pH , reaction time (30 seconds to 40 minutes), temperature  and amount of enzyme , isocitric acid , and dicumarol  on the EROD assay was assessed . Reactions were conducted in triplicate with appropriate enzyme and substrate controls. Results were analyzed at 572 nm as indicated above.CYP1A was only partially purified from liver samples in Cape hake, and phase II metabolic enzymes that are present in solution are known to interfere with the EROD assay . The effSDS-PAGE profiles of the liver fractions  obtainedThe EROD assay was conducted on all fractions obtained from the microsomal preparations to establish which fraction displayed the highest EROD activity. Both spectrophotometric  and fluor = 0.958)  , that isThe NADPH dependence of the EROD assay  indicateμM . Dicumarol (40 μM) was therefore added to EROD reaction mixtures for the duration of this study.  The effect of magnesium sulphate, isocitric acid, and dicumarol on phase II enzymes is demonstrated in Figures M. capensis/M. paradoxus are limited, reports by Goksoyr and Forlin [Merluccius merluccius, reported that the molecular weight of CYP1A was 55 kDa. Most studies present the CYP1A protein as a monomer [SDS-PAGE analysis performed on the fractions obtained from the microsomal preparation procedure  for the d Forlin  have stad Forlin  on the h monomer , 44; theO-deethylase (EROD) assay on each fraction. EROD analysis results for the different fractions  was therefore added to all EROD reaction mixtures for the duration of this study. Although the EROD assay in this particular case was limited by exogenous NADPH supply, other studies using intact fish hepatocytes have demonstrated that the addition of this coenzyme to the overall EROD reaction is not required as the resulting increase in EROD activity was insignificant , 46. TheμM dicumarol to the EROD reaction  that was depleted of this product. Dicumarol was therefore included when performing EROD assays in the present study.A phase II enzyme inhibition study was performed to assess the degree of underestimation of EROD activity during this study. Considering that only partially pure CYP1A samples were analyzed, it is possible that phase II enzymes such as DT-diaphorase and other cytosolic oxidoreductases were present within the mixture. These enzymes may interfere with the EROD assay, as these enzymes have the capacity to further metabolize the product of the reaction, namely, resorufin . The comreaction  showed as et al.  were in μL of enzyme, and a reaction time of 30 seconds. The EROD assay was, however, limited by NADPH supply. The addition of the phase II inhibitor dicumarol significantly increased EROD activity. In conclusion, the EROD assay conditions for Cape hake were optimized in order to provide a method for future studies involving the detection of this pollution biomarker in Cape hake and related species of fish. A few recommendations should be considered for future studies assessing the effect of environmental exposure. All Cape hake samples used in this study arrived frozen which is suitable for this assay optimization study. However, future studies should consider on-site sample preparation (liver excision and nitrogen fixing) as this has been proven to reduce the loss of enzyme activity and variation in individual activity, which could be the result of freezing and thawing of whole fish samples. Future studies could also employ the use of liver samples obtained from fish exposed to AhR agonists as positive controls, in the event fish CYP1A cannot be obtained commercially.In summary, the results presented in this paper demonstrate the development and optimization of an EROD assay for Cape hake. Spectrophotometric analysis at (572 nm) was selected as the preferred detection method for this assay, as a lower degree of variability was obtained between data. Optimum conditions for the EROD assay were found to be as follows: pH of 7.5, temperature of 25°C, 10


Introduction. The objective of this study was to clarify how pitavastatin affects glucoseand lipid metabolism, renal function, and oxidative stress. Methods. Ten Japanesemen (average age of 33.9 years) were orally administered 2 mg of pitavastatin for 4 weeks. Postprandial glucose, lipoprotein metabolism, and oxidative stress markers wereevaluated at 0 and 4 weeks of pitavastatin treatment (2 mg once daily) with a test mealconsisting of total calories: 460 kcal, carbohydrates: 56.5 g , protein: 18 g , lipids: 18 g , and NaCl: 1.6 g. Metabolic parameters were measured at 0, 60, and 120 minutes after test meal ingestion. Results. After administration ofpitavastatin, serum total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, arachidonic acid, insulin, and adjusted urinary excretion of uric acid decreased, whereas creatinine clearance (CCr) and uric acid clearance (CUA) increased. And postprandial versus fasting urine 8-hydroxydeoxyguanosine remained unchanged, while postprandial versus fasting isoprostane decreased after pitavastatin treatment. Next, we compared postprandial glucose and lipid metabolism after test meal ingestion before and after pitavastatin administration. Incremental areas under the curve significantly decreased for triglycerides (P < 0.05) and remnant-like particle cholesterol (P < 0.01), while those for apolipoprotein E (apoE), glucose, insulin, and high-sensitivity C-reactive protein remained unchanged. Conclusion. Pitavastatin improves postprandial oxidative stressalong with hyperlipidemia. It has been generally recognized that postprandial hyperglycemia and hyperlipidemia are highly related to the development of atherosclerosis –5. HyperLipid-lowering drugs, such as statins, fibrates, and ezetimibe are considered to be useful for the treatment of postprandial hyperlipidemia –15. PitaIn addition, there is no previous study on the effect of pitavastatin on oxidative stress markers in postprandial states.8-Hydroxydeoxyguanosine (8-OHdG) and isoprostane are important markers for oxidative stress , 26. IsoWith this background, in this study we investigated the effect of pitavastatin treatment on glucose and lipoprotein metabolism and oxidized stress markers in the postprandial state using a mixed meal consisting of fat, glucose and proteins, and an established test meal  for the n = 10; age 33.9 ± 10.1 years; body height 172.0 ± 4.3 cm; body weight 80.2 ± 25.3 kg; body mass index (BMI) 27.0 ± 8.3 kg/m2; waist circumference 88.5 ± 18.9 cm). None of them had received medication.Japanese men, who agreed to undergo pitavastatin treatment and mixed meal test, were involved in this study , followed by the oral ingestion of a test meal  consisting of total calories 460 kcal, carbohydrates 56.5 g , Protein 18 g , lipids 18 g , and NaCl 1.6 g. The subjects spent 10 min of taking E460F18 with 120 mL of water and underwent blood and urine sampling at 60 minutes and 120 minutes. Pitavastatin administration (2 mg once daily) started from the next day for 4 weeks. After 4 weeks of pitavastatin administration, a meal tolerance test using E460F18 was again conducted exactly the same way it was done before starting pitavastatin.Cholesterol and triglycerides were measured by the enzyme method. HDL-C and LDL-C were measured by direct homogeneous assay methods using detergents. Quantification of remnant-like particles cholesterol (RLP-C) was conducted by a method using an immune-separation technique .μL of plasma was mixed with 300 μL of lipoprotein separation medium consisting of a Sepharose gel suspension to which monoclonal antibodies directed against apoB-100 (JIH) and apoA-1 had been attached. The separation medium was gently mixed for 120 min, and thereafter allowed to settle for another 15 min. The cholesterol content (RLP-C) was assayed enzymatically in the supernatant with an autoanalyzer. Serum Insulin levels were measured using enzyme-linked immunosorbent assay kits . Urinary 8-Hydroxydeoxyguanosine (8-OHdG) and levels were measured using enzyme-linked immunosorbent assay kits . Urinary isoprostane levels were measured using enzyme-linked immunosorbent assay kits .A total of 5 High-sensitivity C-reactive protein (hsCRP) levels were determined using an enzyme-linked immunosorbent assay kit .t-test analysis.Postprandial metabolism was quantified by calculating the incremental area under the curve (iAUC) using each value at 0 minutes, 60 minutes, and 120 minutes after E460F18 loading. Results are presented as mean ± sd. Differences between parameters obtained before and after pitavastatin therapy were evaluated by paired Student's Parameters that did not distribute normally  were analyzed by The Wilcoxon signed-rank test. Dunnett's test was used for the multiple comparisons.This work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.Informed consent was obtained from all of the participants. The institutional review board in Kanazawa Medical University Hospital approved the experimental protocol, and all of the subjects provided informed consent to participate in the study.Ccr, CUA, and R (CUA/Ccr × 100%) and a decrease in adjusted urinary excretion of uric acid.No significant changes were observed in hsCRP and urine 8-OHdG. There were considerable increases in We also analyzed changes in oxidative stress index after pitavastatin treatment. There was no difference in postprandial changes in urine 8-OHdG between before and after pitavastatin treatment, whereas the postprandial increase in urine isoprostanes observed before pitavastatin treatment tended to decrease after pitavastatin treatment .In this study we clarified that pitavastatin treatment produced considerable improvement in markers related to oxidative stress as well as those related to lipid metabolism including RLP-C in postprandial states in Japanese men with abdominal obesity. This is the first study investigating pitavastatin effects on oxidative stress markers in postprandial states after test meal loading as well as fasting states.For fasting states, pitavastatin treatment was associated with significant decreases in fasting insulin levels as well as decreases in fasting TC, LDL-C, and apoB.For postprandial states, pitavastatin treatment was associated with marked reductions in TG-iAUC and more so in RLP-iAUC. Several studies show that plasma RLP-C, not TG, is an independent risk for the development of atherosclerotic disease , 30.There are several studies on the effect of atorvastatin, another strong statin, on postprandial hyperlipidemia and the mechanisms of its improvement have been considered to be an inhibition of the production of very low-density lipoprotein (VLDL) caused by sustained inhibition of cholesterol synthesis in the liver  and incrA postprandial increase in oxidative stress is considered to be an important contributor to the impairment of endothelial function, leading to the development of atherosclerosis and there is a study showing the close link between increases in both TG and that in oxidative stress . Indeed The mechanism by which this occurred has not been clarified yet, but there is a study suggesting the contribution of adiponectin to the reduction of isoprostanes during atorvastatin treatment . Also, pCcr increased after the treatment is compatible with our previous report [We also analyzed the effect of pitavastatin treatment on changes in renal function. Among them the present findings that s report . We press report , since Ns report . These oThe imitations of this study are that the sample size is small and study was done in a single arm. However, we conducted measurements of a wide range of metabolic markers at several time points in postprandial states as well as fasting states before and after pitavastatin treatment. Also it should be noted that to the best of our knowledge there have been only 2 clinical studies on the effect of pitavastatin on postprandial metabolism , 23, botIn conclusion, the present findings suggest that in men with abdominal obesity, pitavastatin improves oxidative stress and renal function along with hyperlipidemia, especially in regards to remnant metabolism, in the postprandial state, contributing to the prevention of the development of atherosclerosis. It is also suggested that pitavastatin improves renal function.


The mechanisms through which exercise reduces cardiovascular disease are not fully understood. We used echocardiograms, cardiac biomarkers and gene expression to investigate cardiovascular effects associated with exercise training.Nineteen sedentary men (22–37 years) completed a 17-week half-marathon training program. Serial measurements of resting heart rate, blood pressure, maximum oxygen consumption, lipids, C-reactive protein, cardiac troponin T, echocardiograms and blood for gene expression were obtained from baseline to peak training. Controls included 22 sedentary men who did not exercise.2 max increased from 37.1 to 42.0 ml/kg/min (p < 0.001). Significant changes were seen in left ventricular wall thickness and mass, stroke volume, resting heart rate and blood pressure (p < 0.001). The control group demonstrated no significant changes. Expression profiling in the training group identified 10 significantly over-expressed and 53 significantly under-expressed loci involved in inflammatory pathways. Dividing the training group into high and low responders based on percent change in VO2 max identified loci that differentiated these two groups at baseline and after training.Among the training group, VOIntensive exercise training leads to significant increase in cardiac and hemodynamic performance, and significant changes in expression of genes involved in immune and inflammatory response. Long-term exercise training has been associated with weight loss, improvements in lipid profile and blood pressure, and cardiac remodeling -3. Despi2 max) less than 50 ml/kg/min, and not previously completed a HM. Twenty-four of the subjects, who were enrolled in a local HM Training Program, were recruited into the training group (TRAIN), and twenty-three male subjects of similar age were recruited into the control group (CON). The CON group did not participate in the training program or undergo any lifestyle modification over the 16 week duration of the study. All subjects volunteered for the study and signed informed consents. TRAIN subjects were compensated for meals, travel time, and training program fees. Exclusion criteria included smoking, known metabolic or cardiovascular conditions (diabetes or dyslipidemia), chronic inflammatory diseases, or chronic use of non-steroidal anti-inflammatory drugs. The protocol was approved by the University of Miami Institutional Review Board for human subjects research.Apparently healthy, untrained and sedentary males (n = 47), between 18 and 45 years of age, participated in the study. Candidates were required to have engaged in less than 2 hours of aerobic training per week prior to enrollment, have an average maximum oxygen consumption  collection. Three subjects were able to complete the peak training data collection, but were unable to complete the HM due to injury. Thus, a total of 19 subjects completed the program with peak training data collected. Of these 19 subjects, 16 subjects completed the 2007 Miami HM and had blood drawn within 30 minutes after the race . Standard measurements were performed according to the American Society of Echocardiography guidelines [Blood was collected in EDTA and standard red top tubes (serum) and processed immediately. Tubes were immediately centrifuged, and the serum was separated and stored in a -80° freezer. Serum was analyzed for cTnT , CRP 1212, and lt-tests were used to compare baseline variable differences between the TRAIN and CON groups. Paired samples t-tests were used to compare changes from baseline to peak training or baseline to post-HM.Clinical and biomarker data were analyzed using SPSS for Windows 15.0 software . Frequency and descriptive statistics were calculated to check all relevant characteristics of the data. Independent samples t-tests to identify statistically significant genes and to measure the strength of relationships between gene expression and a specific response variable. SAM also uses permutation tests to estimate the FDR after accounting for multiple testing corrections [p-values < 0.15 and fold-change greater than 1.5 or less than −1.5 were considered significant. Pathway analysis was conducted using MetaCore™  software.Microarray analysis was performed on blood collected from each subject at baseline and at 16 weeks. Total RNA was extracted and analyzed using the Affymetrix GeneChip Human Gene ST array, which has 28,869 unique genes (or transcripts) which are captured by ~26 probes per gene. Data were normalized using Robust Multi-Array (RMA) Normalization. Significance Analysis of Microarray (SAM) Software  was usedrections . Genes w2 max (rVO2 max) over the course of the training program. Relative VO2 max takes into account and individual’s body weight and is defined as milliliters of oxygen per kilogram of bodyweight per minute. The mean percent change in rVO2 max was calculated and individuals who fell in the upper quartile (upper 25%) of percent change in rVO2 max were labeled as high-responders  and those who fell below the upper quartile were considered low-responders . Gene expression differences between the two groups were studied at baseline and after the 16 weeks of training.Two separate gene expression analyses were performed. The first compared differences in gene expression profiles between the CON and TRAIN subjects before and after the HM training. The second analysis focused only on the TRAIN subjects, by dividing them into high and low responders based on the percent change of their relative VOOf the 16 subjects who completed the HM, the average time to completion was 2:22:21 (H:Min:Sec), SD = 26:33. The fastest time was 1:39:00 and the slowest time was 3:11:58. No runner required medical attention after the race. There were no significant differences between the TRAIN and CON groups on any baseline characteristics other than triglycerides, which were significantly higher (p = 0.03) in the TRAIN group (Table 2 max increased significantly (p = 0.00004) among those in the TRAIN group during the course of the training program. In addition, the following were significantly decreased: (1) resting heart rate (p = 0.002), (2) percent body fat (p = 0.044), and (3) systolic (p = 0.0001) and diastolic (p = 0.007) blood pressure. Average body weight (p = 0.08) and HDL (p = 0.055) decreased slightly (p = 0.08), but no other measured biomarkers  showed significant improvements. There were no significant changes for any variables among the CON subjects  in both groups  showed significant changes   showed significantly elevated expression   were found to be significantly decreased  in the TRAIN group  in high vs. low responders, and one gene showed increased gene expression  in the high compared to low responders group  was most significantly decreased (FC = −2.42). The protein coded by AREG is a member of the epidermal growth factor family. AREG has both growth factor and inhibitory properties . Changes in AREG expression in human neutrophils have been related to exercise in previous studies. For example, following 30 minutes of aerobic exercise, AREG was shown to increase by 3.4 fold [Both TRAIN and CON groups had the same inclusion criteria, which enabled us to maximize the comparison after training. When comparing gene expression profiles in blood collected before training in TRAIN versus CON men, 4 genes showed significant changes in gene expression (FC = (1.5-1.7) FDR < .05) suggesting that the two groups were fairly similar before training. Through pathway analysis, the biological pathways that were significantly up regulated in the TRAIN individuals after training were those primarily involved in the body’s inflammatory and immune response. In looking at the specific genes involved of the 53 genes that showed significantly decreased expression in TRAIN individuals after training, Amphiregulin (3.4 fold . Althoug3.4 fold  examinedDEFA3) also has a role in both the immune and inflammatory response . A previous study linked increased expression of DEFA3 at both the mRNA and protein level to inflammatory diseases [Another gene significantly down-regulated in the TRAIN individuals; defensin, alpha 3, neutrophil-specific (diseases . It is wFGFBP2 (FC = 1.76) and CD160 (FC = 1.55). Both are involved in immune response .When comparing the TRAIN high responders and low responders, several gene expression differences were noted. Two genes that showed increased expression in high responders before training included LRRFIP1 (FC = −1.57). LRRFIP1 has been identified as having a role as both a regulator of toll-like receptor pathway signaling and transcriptional repression. Its expression has been previously shown to be induced by nicotine and proinflammatory cytokines [Of the genes that showed decreased expression in high responders after exercise of particular note is ytokines .AREG, DEFA3, CD160, GPR56, KLRC3, and LRRFIP1. The results presented here are from a single sample and do not withstand conservative corrections for multiple comparisons. The study’s sample is limited to men and therefore cannot necessarily be generalized to the broader population which could include females However, they generate interesting hypotheses about a possible role of genes involved in immunity and inflammation in responding to intensive exercise training, which bear examination in other data sets.In summary, in previously sedentary individuals, intensive training for a HM over 17 weeks leads to increased cardiac and hemodynamic performance that is not associated with longitudinal changes in known cardiac biomarkers. Elevated cTnT occurred in only 1 of 16 subjects who completed the HM. Training is also associated with changes in blood gene expression that reflects the anabolic state of the body, as well as changes in the body’s immune and inflammatory response that could be more sensitive precursors to changes in serum biomarkers. Whereas some genes involved in the immune/inflammatory response showed increased expression, others showed decreased expression implying that exercise brings about changes that both suppress aspects of the immune system while stimulating others. Genes with changes in gene expression as response to HM-training include The authors declare that they have no competing interests.DDV: study design, RNA extraction, gene expression and pathway analysis, manuscript preparation. GC: echocardiogram data collection, cardiac biomarker analysis, manuscript preparation. MS: ascertainment, data collection. RJM: study design, cardiac biomarker analysis, manuscript editing. KJ: ascertainment, data collection. LN: microarray and gene expression analysis. AP: data collection, study design, editing. DS: study design, gene expression analysis, manuscript editing. PJGC: study design, manuscript editing. ER statistical analysis, gene expression and pathway analysis, manuscript editing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2052-1847/6/6/prepub


In patients with left ventricular hypertrophy (LVH), LV midwall fractional shortening (FS) is used as a measure of LV systolic performance that is more physiologically appropriate than conventional FS. For evaluation of LV volume and ejection fraction (EF), 2-dimensional (2D) echocardiography is more accurate than M-mode echocardiography. The purpose of this study was to assess systolic performance by midwall EF using 2D speckle tracking echocardiography (STE).Sixty patients were enrolled in the study. Patients were divided into two groups with LVH (n = 30) and without LVH . LV systolic function was compared between the two groups and the relationships of left ventricular mass index (LVMI) with LV systolic parameters, including midwall EF, were investigated.Midwall EF in the LVH group was significantly lower than that in the control group . Midwall FS was also significantly lower in the LVH group , but EF did not differ significantly between the two groups. There were significant correlations between midwall EF and LVMI  and between midwall FS and LVMI , with midwall EF having the higher correlation.These results show that midwall EF can be determined using 2D STE. Midwall EF can be used to monitor LV systolic dysfunction, which is not possible with conventional EF. Evaluation of midwall EF may allow assessment of new parameters of LV systolic function in patients with LV geometric variability. Left ventricular (LV) systolic performance is often assessed by ejection fraction (EF) and fractional shortening (FS). It is said that despite depression of LV systolic function, LVEF and LVFS are preserved in patients with left ventricular hypertrophy (LVH) -6. Howev2 for women and >114 g/m2 for men. The causative of patients with LVH was essential hypertension with the exception of chronic renal failure, idiopathic hypertrophic cardiomyopathy, amyloidosis. Patients with myocardial infarction, atrial fibrillation, valvular disorders, and any other structural heart disease were excluded. Patients with poor echocardiographic image quality in the apical 2-chamber and 4-chamber views were also excluded. LV function was measured using conventional echocardiography, tissue Doppler imaging (TDI), and 2D-STE. LV systolic function was assessed by EF, midwall FS, midwall EF, and longitudinal strain. Systolic function was compared between the two groups and the relationships of LVMI with LV systolic parameters, including midwall EF, were investigated. Ethical review board approval from our hospital was obtained.Sixty patients were enrolled in the study. Patients were divided into two groups with LVH (n = 30) and without LVH . LVH was defined as an LV mass index (LVMI) >96 g/mEchocardiographic studies were performed using commercial equipment  with the patient in the left lateral decubitus position. Images were obtained using a 3.5-4.0 MHz transducer in the parasternal short-axis and apical 4-chamber views . InterveMidwall FS = (LVIDd + Hd/2) − (LVIDs + Hs/2)/(LVIDd + Hd/2).1. LVIDd + Hd/2)3 − LVIDd3 = (LVIDs + Hs/2)3 − LVIDs3.2. Volume of myocardium between the midwall and the endocardium = .where LVID is the LV internal dimension, TDI was performed in all patients with images taken based on the guidelines of the American Society of Echocardiography . Using t2D image analysis was performed on digitally stored images . Real-time 2D data sets were obtained from the apical 2-chamber and 4-chamber images. Images were recorded to allow for reliable operation of the software .Myocardial longitudinal strain measurement was assessed on 4-chamber image with speckle tracking analysis. The traced endcardium is automatically divided into six segments; septal, anteroseptal, anterior, lateral, posterior, and inferior. The average peak strain measured in the longitudinal directions defined as the longitudinal strain.The midwall EF measurements were obtained using semiautomated speckle tracking . Image acquisition was performed with end-expiratory breath holding to reduce the scattering of values. One cardiac cycle was analyzed in each patient. The position of the midwall was determined using the landmark of the midpoint between the epicardial and endocardial borders depending on the LV wall thickness. Anatomic landmarks, including these midpoints and a point on the apical endocardium, were manually initialized at the end of diastole only. Following this initialization, the software automatically positioned sixteen regions of interests (ROI) on the midwall LV cavity surface. In systole, the initial ROI was not positioned at the midpoint of the wall thickness because systolic thickening of the inner layer is larger than that of the outer layer. Further manual adjustments of the position of the ROI in the end-diastolic frame were performed as necessary . A repreThese data were obtained using the same method as that for midwall EF measurement, except for the positions of ROIs, which were based on anatomic landmarks, including septal and lateral points on the mitral annulus and a point on the apical endocardium. The EF was then calculated from apical 4-chamber and 2-chamber views, as for the midwall EF in 2D STE.t-test was used to compare continuous variables and a χ2 test was used for categorical variables. Simple linear regression analysis was used to evaluate relationships between variables of interest, with p < 0.05 considered to indicate significance.Data are expressed as means ± SD. A Student Of the 69 patients who were initially recruited, 9 were excluded because the echocardiographic image quality was unsuitable for quantitative 2D STE analysis. Thus, 60 patients were subsequently enrolled in the study. Patients were divided into two groups with LVH (n = 30) and without LVH . The characteristics of the patients in these groups are shown in Table 2 vs. 86.9±10.8 g/m2, p <0.0001) compared to the respective values in the control group. LVDd, LVDs, LVFS, E velocity, A velocity, and E/A did not differ between the two groups.Echo parameters in the LVH and control groups are shown in Table Systolic echo and TDI parameters are shown in Table Relationships between echo parameters and LVMI are shown in Table The reproducibility of the EF and midwall EF measurements was evaluated by calculating the intra- and interobserver variability in 30 of the 60 patients selected at random. The intra- and interobserver variabilities were 4.1±3.3% and 4.8±3.2%, respectively, for EF, and 6.1±4.4% and 6.4±4.2%, respectively, for midwall EF.The midwall EF in the LVH group was significantly lower than that in the control group and midwall EF was correlated with the degree of LVH. Our study showed the utility of midwall EF for assessing systolic performance of the hypertrophic left ventricle. Also, this method may be clinically useful and is likely to have low observer variability. As far as we are aware, there has only been one previous study of midwall EF. Jung et al. found that the midwall EF also discriminates the systolic function between patients with LVH and normal subjects , but theLVH is an independent predictor of adverse cardiovascular events in hypertension ,15. AccuMidwall FS has been used to detect depressed LV systolic function in patients with LVH ,3-5. A pThe correlation of LVMI with midwall EF was higher than with TDI parameters. It has been suggested that TDI can be used to quantify regional ventricular function objectively and the mitral annular velocity may be a more sensitive index of LV function . HoweverOur study also showed the usefulness of 2D STE for measurement of midwall EF. The STE technique relies on tracking of natural acoustic markers in the myocardium from frame to frame throughout the cardiac cycle using a sum of absolute differences algorithm . Thus, tIn this study, midwall EF correlated with the degree of LVH. Patients with LVH have intrinsic dysfunction in both systole and diastole. Our study showed that midwall EF can detect LV systolic dysfunction, which cannot be detected by conventional EF. This may be an important sign of LV dysfunction in patients with hypertension, which may not always be due to diastolic dysfunction, but can often be caused by systolic dysfunction, as assessed by midwall EF . EvaluatOur methods demonstrated that the midwall EF is clinically useful for detecting the systolic function in addition to midwall FS, TDI, and strain. Midwall EF can detect LV systolic dysfunction, which cannot be detected by conventional EF. The intrinsic systolic dysfunction may affect predictive of subsequent morbidity and mortality in patients with LVH. Midwall EF will have possibilities to detect the beneficial change of intrinsic systolic dysfunction by medical treatment in clinical settings.The current study has several limitations. First, we did not examine the influence of afterload, which may affect the midwall analysis. Second, the subjects did not constitute a consecutive series and were selected according to image quality. Finally, midwall EF using 2D STE is not theoretically the same as conventional midwall FS, which is needed to measure both systole and diastole.The study showed that midwall EF can be evaluated using 2D STE. The midwall EF in the LVH group was significantly lower than that in the control group and midwall EF was correlated with the degree of LVH. This measurement can be used to detect LV systolic dysfunction, which cannot be detected by conventional EF. Thus, evaluation of midwall EF may allow assessment of new parameters of LV systolic function in patients with various LV geometries.LVH: Left ventricular hypertrophy; FS: Fractional shortening; EF: Ejection fraction; 2D: 2-dimensional; STE: Speckle tracking echocardiography; LVMI: Left ventricular mass index; TDI: Tissue Doppler imaging; IVST: Interventricular septal thickness; PWT: Posterior wall thickness; LVDd: Left ventricular end-diastolic diameter; LVDs: Left ventricular end-systolic diameter; LAD: Left atrial dimension; SV: Stroke volume; DCT: Deceleration time; E velocity: Peak early mitral flow velocity; A velocity: Peak late mitral flow velocity; E/A: Ratio of mitral E and A; S’: The early systolic mitral annular velocity; E’: The early diastolic mitral annular velocity; A’: The late diastolic annular velocity; ROI: Regions of interests.The authors have no competing interests.HY, MS, and GH planned the study, investigated all patients, performed measurements and analyzed the data. YK and TO analyzed data and wrote the manuscript. MN and KS made critical review of the paper. All authors read and approved the final manuscript.


Background. Low body mass index (BMI) at antiretroviral therapy (ART) initiation is associated with early mortality, but the etiology is not well understood. We hypothesized that low pretreatment serum phosphate, a critical cellular metabolism intermediate primarily stored in skeletal muscle, may predict mortality within the first 12 weeks of ART. Methods. We prospectively studied 352 HIV-infected adults initiating ART in Lusaka, Zambia to estimate the odds of death for each 0.1 mmol/L decrease in baseline phosphate after adjusting for established predictors of mortality. Results. The distribution of phosphate values was similar across BMI categories . Among the 145 participants with BMI <18.5 kg/m2, 28 (19%) died within 12 weeks. Lower pretreatment serum phosphate was associated with increased mortality (odds ratio (OR) 1.24 per 0.1 mmol/L decrement, 95% CI: 1.05 to 1.47; P = 0.01) after adjusting for sex, age, and CD4+ lymphocyte count. A similar relationship was not observed among participants with BMI ≥18.5 kg/m2 . Conclusions. The association of low pretreatment serum phosphate level and early ART mortality among undernourished individuals may represent a variant of the refeeding syndrome. Further studies of cellular metabolism in this population are needed. While armal BMI , the losrmal BMI –9.refeeding syndrome [Skeletal muscle mass, a major reservoir of bioavailable phosphate, is reduced early in HIV-associated weight loss, and among HIV-uninfected persons chronic undernutrition is associated with a reduction in mitochondrial enzyme activity which rapidly normalizes with nutritional support –12. Priosyndrome –16. We hsyndrome , 18. To + lymphocyte count <200 cells/μL, or WHO stage 3 disease and a CD4+ lymphocyte count <350 cells/μL), intention to start therapy the same day and remain in the area for the study duration, and agreement to adhere to the additional study visits and laboratory testing requirements. One cohort (NEMART) [2 or CD4+ lymphocyte count <50 cells/μL), and the other (DGPLEAD) [2 and CD4+ ≥50 cells/μL. All screened patients offered the opportunity to enroll in NEMART or DGPLEAD elected to participate in the study. In this analysis we combine data from both cohorts to evaluate the common primary study endpoint of all-cause mortality within the first 12 weeks of ART. Between November 2006 and December 2007, we enrolled HIV-infected adults starting ART at a single public sector primary care clinic in Lusaka, Zambia into two simultaneous observational cohorts to evaluate metabolic predictors of mortality within 12 weeks of treatment initiation. Both cohorts have been described previously –21. Enro(NEMART)  compriseStudy participants were evaluated at enrollment by a research nurse and a clinical officer, and additional assessment was performed by a supervising physician as needed. The initial visit included a detailed health history, review of systems, physical examination, and laboratory testing ). The initial ART regimen was selected from the national program formulary: lamivudine, efavirenz or nevirapine, and zidovudine or stavudine. In July 2007, tenofovir with emtricitabine replaced lamivudine, zidovudine, and stavudine in the national program's first-line regimen . Participants in the NEMART cohort were evaluated by the study staff at 1, 2, 4, 8, and 12 weeks after initiating ART. Serum electrolyte measurements were done in real time, and when deficiencies of phosphate were detected participants were given supplements within seven days according to a predetermined algorithm based on serum levels. Participants in the DGPLEAD cohort were evaluated by study staff at baseline and after 12 weeks of ART; their serum samples were frozen at −80°F and assayed after the study was completed. + lymphocyte counts were performed using a Beckman Coulter Epics XL-MCL flow cytometer . Chemistry assays were measured using a Roche COBAS Integra 400+  or a Pointe 180 Chemistry Analyzer . CD4If a participant missed a study visit and could not be reached by mobile phone, community outreach teams attempted to locate the patient using housing locator forms completed at enrollment . If the 2) [+ lymphocyte count at ART initiation and secondary analyses further adjusted for baseline albumin, ferritin, and hsCRP, and concomitant tuberculosis treatment. Patients lost to follow-up were excluded from primary analyses; a secondary analysis considered the combined endpoint of lost to follow-up or death. R-software 2.9.2 (http://www.r-project.org/) was used for data analyses.We used logistic regression to assess the relationship between a 0.1 mmol/L decrease in baseline serum phosphate and the odds of death within 12 weeks of ART in the full cohort (including an interaction term for BMI and phosphate) and after stratifying the cohort according to WHO BMI criteria for grading malnutrition  . All modThe study protocol was approved by the University of Zambia Research Ethics Committee and the Institutional Review Boards at the University of Alabama at Birmingham and Vanderbilt University. All procedures were consistent with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. All participants provided written consent.2 died compared to 12 (7%) of those with BMI ≥18.5 kg/m2. The alive, deceased, and lost to follow-up groups differed by median age, BMI, CD4+ lymphocyte count, and serum phosphate, ferritin, and hsCRP levels . There was no difference between the three groups in the proportion of participants receiving tuberculosis treatment at the time of ART initiation. Among those with known vital status at 12 weeks, deceased participants had significantly lower BMI, CD4+ lymphocyte counts, serum albumin levels, and serum phosphate levels, but higher inflammation biomarkers (serum hsCRP and ferritin), compared to survivors .Vital status was available for 307 of 352 enrolled participants at 12 weeks (87%) and 45 were classified as lost to follow-up; 28 (19%) of those with pretreatment BMI <18.5 kg/m+ lymphocyte count (36 cells/μL), which was closer to the median of 50 cells/μL observed in the deceased group versus the 109 cells/μL in the alive group. However, median serum levels of phosphate, albumin, ferritin, and hsCRP among the lost participants more closely approximated the alive group compared to the deceased. Participants lost to follow-up prior to 12 weeks were more likely to be female and had a lower median age, but the clinical characteristics of this group did not consistently resemble either the alive or deceased participants. Lost participants had the lowest median CD4P = 0.41), indicating that low phosphate values were not clustered among the low-BMI participants  [P = 0.20). While tenofovir has been linked to renal phosphate wasting and toxicity in some reports, though not in randomized trials, the initiation of a tenofovir-containing regimen in our cohort was not associated with increased mortality (P = 0.12) [Baseline phosphate values were generally uniform across the BMI range  . At week = 0.12) –28.+ lymphocyte count, both serum phosphate (odds ratio (OR) 1.18 per 0.1 mmol/L decrease; P = 0.02) and BMI  were significantly associated with 12-week mortality. We did not detect an interaction of BMI and phosphate on the log-odds of mortality (P = 0.52). Participants with BMI <18.5 kg/m2  had an increased odds of morality  for each 0.1 mmol/L decrease in baseline phosphate. A similar relationship was observed among those with BMI <17 kg/m2  (2 (n = 49) the relationship between serum phosphate and outcome was not statistically significant , though the death rate was substantially higher (27%). Among participants with BMI ≥18.5 kg/m2 (n = 161) we did not detect an association between baseline phosphate and 12-week mortality . In a model including all patients but dichotomizing BMI at <18.5 versus ≥18.5, the interaction between BMI and phosphate was significant (P = 0.046).In a logistic regression model excluding participants with unknown vital status and adjusted for sex, age, and CD4 = 0.03) . Among t2. Similarly, the relationship between serum phosphate and mortality among those with BMI <18.5 kg/m2 remained significant after adjusting for concomitant tuberculosis treatment , and TB treatment was not associated with mortality in the other models. When the logistic regression model was further adjusted for pretreatment serum hsCRP, ferritin, and albumin levels, there was minimal change in the odds of mortality with each 0.1 mmol/L reduction in baseline phosphate among those with BMI <18.5 kg/mP = 0.76) or those with a BMI <18.5 kg/m2 . We attribute this lack of an association to likely heterogeneity in both the clinical characteristics and 12 week outcome among participants lost to follow-up.When we repeated the analyses using the combined endpoint of death or loss to follow-up at 12 weeks, there was no significant association between a 0.1 mmol/L decrease in pretreatment serum phosphate and this outcome among all participants . Additionally, plasma HIV-1 viral load measurements were not utilized in routine clinical care at the time of the study, which may have introduced unmeasured confounding. The criteria for classifying a participant as lost to follow-up in our 12-week study differed from the definition used by the Zambian National ART program for pragmatic reasons. At the time of the study, patients in the national program were classified as lost to follow-up if they did not return to clinic within 37 days after a scheduled pharmacy visit or, if no pharmacy visit was scheduled, 60 days after the last clinical visit. This definition would not accurately classify participants at a single time point , and therefore a more intensive outreach procedure utilizing home locator forms and phone calls was used to trace study participants. While some participants classified as lost may have eventually returned to care, the follow-up rates observed in our study approximated rates in the Zambian National ART program and in similar resource-limited settings .We hypothesize that the observed association between lower pretreatment serum phosphate and early ART mortality may represent a variant of the refeeding syndrome associated with advanced HIV disease, in which depleted reserves of bioavailable phosphate  are insufficient to maintain electrolyte homeostasis in response to a rise in phosphate-dependent cellular respiration after treatment initiation. The increased metabolic activity could result from viral suppression, immune reconstitution, or increased tissue repair and redeposition following a decline in inflammation-induced catabolism, possibly in combination with increased physical activity and dietary intake. Under these circumstances, low-BMI individuals with poor reserves of muscle mass may not be able to mobilize sufficient phosphate to meet metabolic demands. The consequences of a failure to maintain adequate bioavailable phosphate after ART initiation would likely mimic a classic refeeding syndrome, in which potentially lethal cardiovascular, respiratory, and neurologic sequelae result from electrolyte and fluid shifts accompany a reversal from catabolism and fat oxidation to the utilization of exogenous carbohydrate , 30–32. At present, a role for refeeding syndrome in early ART mortality is provisional and additional studies are needed to understand whether the pathophysiologic processes contributing to death among malnourished individuals with low serum phosphate are consistent with this syndrome or a separate disorder. Further investigation of phosphate-dependent biological processes in these patients, including mitochondrial protein content and function, intracellular phosphate content, and potassium and magnesium homeostasis, will be critical for identifying the specific biological mechanisms involved and designing effective treatments to reduce mortality in the early ART period.+ lymphocyte count, serum albumin, and biomarkers of inflammation. The effect was not seen among individuals starting ART with normal BMI and may reflect an acute physiologic dysfunction resulting from insufficient bioavailable phosphate reserves in the immediate posttreatment period among individuals with reduced metabolically active tissue. Given the high early mortality rates among low BMI adults starting ART in Africa, further studies of metabolic dysfunction in this population are warranted to confirm these findings and explore potential mechanisms. Additionally, clinical trials to explore the benefits and risks of phosphate supplementation on ART outcomes among low-BMI individuals are needed.Low pretreatment serum phosphate levels were associated with increased risk for early mortality among adults with low BMI starting ART in Zambia. This association was independent of established risk factors for early ART mortality, including BMI, CD4