LT4/LT3 Combination Therapy vs. Monotherapy with LT4 for Persistent Symptoms of Hypothyroidism: A Systematic Review.

Regardless of the cause, hypothyroidism should be treated with levothyroxine. The objectives of management are the normalization of TSH levels and the relief of symptoms. In general, the vast majority of patients who achieve normalization of TSH levels show a resolution of symptoms; however, for a small number of individuals, symptoms persist (despite adequate control of TSH). This scenario generates a dilemma in the therapeutic approach to these patients, because even when excluding other causes or concomitant diseases that can explain the persistence of symptoms, pharmacological management strategies are scarce. Consequently, the efficacy of some less conventional approaches to therapy, such as the use of LT3 monotherapy, desiccated thyroid extracts, and LT4/LT3 combinations, in addressing persistent hypothyroid symptoms have been evaluated in multiple studies. The majority of these studies did not observe a significant benefit from these "nonconventional" therapies in comparison to results with LT4 monotherapy alone. Nevertheless, some studies report that a significant proportion of patients prefer an alternative to monotherapy with LT4. The most common approach has been to prescribe a combination of LT4 and LT3, and this review describes and analyzes the current evidence of the efficacy of LT4/LT3 combination therapy vs. LT4 monotherapy in addressing persistent hypothyroidism symptoms to provide suggested guidelines for clinicians in the management of these patients.

Association of Free Thyroxine with Progression-Free Survival in Intermediate and High Risk Differentiated Thyroid Cancer.

CONTEXT: Supraphysiologic thyroxine (T4) doses are used in intermediate and high-risk patients with differentiated thyroid cancer (IR/HR-DTC) to suppress tumor progression by thyrotropin (TSH). However, preclinical data suggest that T4 can also act as a growth stimulus for cancer, but there is no clinical evidence supporting this claim. OBJECTIVE: We analyzed the association between free T4 (FT4) and progression-free survival (PFS) in patients with IR/HR-DTC. METHODS: This longitudinal cohort study, approved by multi-institutional review board, included patients with IR/HR-DTC treated uniformly with total thyroidectomy, radioiodine (RAI), and TSH suppression therapy, with at least three TSH and FT4 values available. Association between FT4 and PFS at landmarks 6, 12, and 18 months was assessed by Kaplan-Meier survival curves, while competing risks were assessed through Cox proportional hazards model. RESULTS: From 739 screened patients 382 met the inclusion criteria and were characterized by a median age of 46 (34-59) years, 64.1% women, treated with a median RAI dosage of 159 (110-410) mCi. During follow up of 7.1 (3.4-12.7) years 34.6% experienced disease progression.Elevated FT4, observed in 29.3% of patients, was not associated with worse PFS (HR 0.9, CI 0.54-1.5, p=0.69), while age (HR 1.02, CI 1.004-1.04, p=0.01), tumor size (HR 1.15, CI 1.04-1.28, p=0.01), and metastases to the lateral neck lymph nodes (HR 2.9, CI 1.7-4.74, p<0.001), bones (HR 4.87, CI 1.79-13.3, p=0.002), and brain (HR 5.56, CI 2.54-12.2, p<0.001) were associated with shorter PFS. CONCLUSIONS: Contrary to preclinical evidence, elevated FT4 levels do not affect PFS in patients with IR/HR-DTC.

Sex-Specific Expression of Histone Lysine Demethylases (KDMs) in Thyroid Cancer.

BACKGROUND: The incidence of thyroid cancer in women is 3-4-fold higher than in men. To characterize sex-specific molecular alterations in thyroid cancer, we examined the expression of sex-biased genes in normal thyroids and thyroid tumors. METHODS: Ingenuity pathways analysis was used to define sex-biased gene networks using data from the Cancer Genome Atlas (TCGA). Confirmatory studies were performed through the analysis of histone lysine demethylases (KDMs) expression by real-time PCR and immunostaining. RESULTS: In normal thyroids, 44 sex-biased genes were comparatively upregulated in male and 28 in female patients. The expressions of 37/72 (51%) sex-biased genes were affected in cancer tissues compared with normal thyroids. Gene network analyses revealed sex-specific patterns in the expressions of KDM5C, KDM5D, and KDM6A. In confirmatory studies, KDM5D mRNA and protein were detected only in males, whereas KDM5C and KDM6A were detected in samples from male and female patients. Nuclear staining with anti-KDMs was found in normal thyroids, but a loss of nuclear expression with a concomitant gain of cytoplasmic staining was observed in cancer tissues. CONCLUSIONS: Normal thyroids have a sex-specific molecular signature, and the development of thyroid cancer is associated with a differential expression of sex-biased genes. The sex-specific expression of KDMs, coupled with cancer-related alterations in their intracellular localization, may contribute to mechanisms underlying sex differences in thyroid tumorigenesis.

Improved sexual function after parathyroidectomy in women with primary hyperparathyroidism.

BACKGROUND: The study aimed to evaluate whether women with primary hyperparathyroidism (PHPT) experience improvement in their sexual function after parathyroidectomy. METHODS: Women with PHPT or benign thyroid nodules (controls) undergoing surgery were administered the validated Parathyroidectomy Assessment Score (PAS), Patient-Reported Outcomes Measurement Information System-29 (PROMIS-29) and Female Sexual Function Index (FSFI) pre-operatively, at 3 months and 6 months postoperatively. RESULTS: Of the 26 PHPT and 18 control patients, PHPT patients were older (53.1 vs 45.3 years, p â€‹= â€‹0.008). Post-operatively, both PHPT (pre-op 2.4 vs 3-month 3.0 vs 6-month 2.4, p â€‹= â€‹0.022) and control patients (pre-operative 2.4 vs 3-month 3.3 vs 6-month 3.6, p â€‹= â€‹0.032) reported increased desire for sexual activities. In addition, PHPT patients experienced increased arousal (pre-operative 2.7 vs 3-month 3.9 vs 6-month 3.6, p â€‹= â€‹0.047) and satisfaction (pre-operative 3.0 vs 3-month 4.8 vs 6-month 4.0, p â€‹= â€‹0.006). CONCLUSIONS: The current study indicates that women with PHPT may experience improved sexual function after parathyroidectomy.

Efficacy of Levothyroxine Sodium Soft Gelatin Capsules in Thyroidectomized Patients Taking Proton Pump Inhibitors: An Open-Label Study.

Background: Treatment with proton pump inhibitors (PPIs) and antacids affects the gastrointestinal absorption of levothyroxine sodium (LT4) tablets. Patients with hypothyroidism taking LT4 and PPIs or antacids, thus, require appropriate monitoring. The objective of this study was to determine whether a soft gelatin capsule of LT4 (Tirosint®) would obviate the effect of PPIs on LT4 absorption. The objective was achieved by assessing the effects of a switch from a conventional LT4 tablet form to the same dose as soft capsules in thyroidectomized patients on treatment with LT4 and PPIs. Methods: Patients with history of hypothyroidism due to total thyroidectomy on stable treatment with LT4 tablets, and with gastrointestinal disease treated with PPIs, were switched to a 12-week treatment with Tirosint at the same dose of the LT4 tablets, while maintaining treatment with PPIs. Serum thyrotropin (TSH) levels were the primary endpoint of the study. Secondary efficacy endpoints were: serum levels of free thyroxine (fT4), total thyroxine (TT4), free triiodothyronine (fT3), total triiodothyronine (TT3), creatine-phosphokinase (CPK), sex-hormone binding globulin, ferritin, angiotensin converting enzyme, and a lipid panel. Results: Forty-seven patients (36 females and 11 males, mean age 55.4 years) were enrolled and 45 of them completed the study (2 patients withdrew consent). During treatment with Tirosint, mean TSH levels demonstrated a statistically significant decrease (mean changes from baseline: -0.32 mIU/L at week 6 and -0.68 mIU/L at week 12) and concomitant increases in thyroid hormone (TH) levels from baseline to week 12, which were statistically significant for fT3 and TT3 (mean changes from baseline: 0.26 pmol/L and 0.10 nmol/L, respectively). Significant decreases of serum low-density lipoprotein, total cholesterol, and CPK levels were observed at week 12. No signs/symptoms arose during the study that could be specifically correlated to either hypo- or hyperthyroidism. Conclusions: In thyroidectomized patients taking PPIs and replacement LT4, a switch from conventional LT4 tablets to LT4 soft capsules at the same dose was associated with a significant decrease in TSH and increase in TH, indicating that LT4 absorption may be less affected by PPIs when given in the form of soft capsules. Clinical Trial Registration: NCT03094416.

Efficacy of machine learning to identify clinical factors influencing levothyroxine dosage after total thyroidectomy.

BACKGROUND: We employed Machine Learning (ML) to evaluate potential additional clinical factors influencing replacement dosage requirements of levothyroxine. METHOD: This was a retrospective study of patients who underwent total or completion thyroidectomy with benign pathology. Patients who achieved an euthyroid state were included in three different ML models. RESULTS: Of the 487 patients included, mean age was 54.1 ± 14.1 years, 86.0% were females, 39.0% were White, 53.0% Black, 2.7% Hispanic, 1.4% Asian, and 3.9% Other. The Extreme Gradient Boosting (XGBoost) model achieved the highest accuracy at 61.0% in predicting adequate dosage compared to 47.0% based on 1.6 mcg/kg/day (p < 0.05). The Poisson regression indicated non-Caucasian race (p < 0.05), routine alcohol use (estimate = 0.03, p = 0.02), and osteoarthritis (estimate = -0.10, p < 0.001) in addition to known factors such as age (estimate = -0.003, p < 0.001), sex (female, estimate = -0.06, p < 0.001), and weight (estimate = 0.01, p < 0.001) were associated with the dosing of levothyroxine. CONCLUSIONS: Along with weight, sex, age, and BMI, ML algorithms indicated that race, ethnicity, lifestyle and comorbidity factors also may impact levothyroxine dosing in post-thyroidectomy patients with benign conditions.

Clinical Factors Predictive of Lymph Node Metastasis in Thyroid Cancer Patients: A Multivariate Analysis.

BACKGROUND: Early-stage thyroid cancers have excellent survival. However, lymph node metastases (LNM) confer a worse prognosis and are not always known preoperatively. Therefore, investigation on the clinical and histological factors predictive of LNM in thyroid cancers was conducted to tailor the extent of surgery and radioactive iodine therapy. STUDY DESIGN: Multivariate logistic regressions were performed based on retrospective data from thyroid cancer patients seen between 2013 and 2020 at a single institution. RESULTS: Among 913 patients, mean age was 49.4 years, 76.5% were female, 58.3% were White, 21.2% were Black, and 27.9% had LNM. In the multivariate analyses in which the outcome was LNM, White (odds ratio [OR] 1.74, 95% CI 0.98 to 3.15, p = 0.064) and Hispanic patients (OR 2.36, 95% CI 0.97 to 5.77, p = 0.059) trended toward higher risk of LNM compared to Black patients, whereas age (OR 0.98, 95% CI 0.97 to 1.00, p = 0.008) showed protective effect. Tumor size (OR 1.04, 95% CI 1.01 to 1.07, p = 0.007), extrathyroidal extension (OR 2.46, 95% CI 1.53 to 3.97, p < 0.001), lymphovascular invasion (OR 6.30, 95% CI 3.68 to 11.14, p < 0.001), and multifocality (OR 1.47, 95% CI 1.01 to 2.12, p = 0.042) were associated with higher risk of LNM. In another model with outcome as >5 LNM, tumor size (OR 1.07, 95% CI 1.03 to 1.11, p = 0.001), age (OR 0.95, 95% CI 0.93 to 0.97, p < 0.001), extrathyroidal extension (OR 3.20, 95% CI 1.83 to 5.61, p < 0.001), and lymphovascular invasion (OR 6.82, 95% CI 3.87 to 12.17, p < 0.001) remained significant predictors. CONCLUSION: Our analyses demonstrated and confirmed that age, tumor size, extrathyroidal extension, and lymphovascular invasion are independent predictors of significant LNM, thereby conferring higher risk of recurrence. Risk of LNM based on these patient characteristics should be considered when planning an operative approach.

Hashimoto thyroiditis: an evidence-based guide to etiology, diagnosis and treatment.

Hashimoto thyroiditis (HT) is a common autoimmune disorder, affecting women 7-10 times more often than men, that develops because of genetic susceptibility, Xchromosome inactivation patterns modulated by environmental factors as well as microbiome composition, and leads to an imbalance in self­tolerance mechanisms. The consequential thyroid infiltration by lymphocytes, potentiated by antibody­mediated autoimmune response through the antibodies against thyroid peroxidase (TPOAbs), leads to a destruction of thyrocytes. The presence of TPOAbs is associated with a 2 to 4­fold increase in the risk of recurrent miscarriages and preterm birth in pregnant women. The clinical presentation of HT includes: (A) thyrotoxicosis, when stored thyroid hormones are released to circulation from destroyed thyroid follicles; (B) euthyroidism, when preserved thyroid tissue compensates for destroyed thyrocytes; and (C) hypothyroidism, when thyroid hormone production by the affected thyroid gland is insufficient. The management of Hashitoxicosis is based on symptoms control usually with ß­blockers, euthyroidism requires periodical thyroid stimulating hormone measurements to assess for progression to hypothyroidism, and hypothyroidism is treated with thyroid hormone replacement therapy. The dose of levothyroxine (LT4) used for treatment is based on the degree of preserved thyroid functionality and lean body mass, and usually ranges from 1.4 to 1.8 mcg/kg/day. There is insufficient evidence to recommend for or against therapy with triiodothyronine (T3), apart from in pregnancy when only levothyroxine is indicated, as T3 does not sufficiently cross fetal blood­brain barrier. HT is associated with 1.6 times higher risk of papillary thyroid cancer and 60 times higher risk of thyroid lymphoma than in general the population.

Metastatic Differentiated Thyroid Cancer Survival Is Unaffected by Mode of Preparation for 131I Administration.

Context: Recombinant human thyrotropin (rhTSH) is currently not Food and Drug Administration approved for the treatment of high-risk patients with differentiated thyroid cancer (DTC). Objective: The goal of our study was to compare the outcomes in higher-risk patients with metastatic DTC prepared for radioiodine (RAI) therapy with rhTSH vs thyroid hormone withdrawal (THW). Methods: A retrospective chart review was performed of patients with metastatic DTC in follow-up at MedStar Washington Hospital Center and MedStar Georgetown University Hospital from 2009 to 2017. Patients were divided according to their preparation for RAI therapy, with assessment of progression-free survival (PFS) and overall survival (OS). Results: Fifty-five patients with distant metastases (16 men, 39 women) were prepared for RAI therapy exclusively either with rhTSH (n = 27) or with THW (n = 28). There were no statistically significant differences between the groups regarding clinicopathological features and history of RAI therapies. The median follow-up time for patients with rhTSH-aided therapies was 4.2 years (range, 3.3-5.5 years) and for patients with THW-aided therapies was 6.8 years (range, 4.2-11.6 years) (P = .002). Multivariate analysis showed that the method of thyrotropin stimulation was not associated with a difference in PFS or OS. Conclusion: As has been shown previously for low-risk DTC, this study indicates that the mode of preparation for RAI therapy does not appear to influence the outcomes of patients with metastatic DTC. PFS and OS were similar for patients with THW-aided or rhTSH-aided RAI therapies.

Sex Differences in Differentiated Thyroid Cancer.

Background: Sex dimorphism strongly impacts tumor biology, with most cancers having a male predominance. Uniquely, thyroid cancer (TC) is the only nonreproductive cancer with striking female predominance with three- to four-fold higher incidence among females, although males generally have more aggressive disease. The molecular basis for this observation is not known, and current approaches in treatment and surveillance are not sex specific. Summary: Although TC has overall good prognosis, 6-20% of patients develop regional or distant metastasis, one third of whom are not responsive to conventional treatment approaches and suffer a 10-year survival rate of only 10%. More efficacious treatment strategies are needed for these aggressive TCs, as tyrosine kinase inhibitors and immunotherapy have major toxicities without demonstrable overall survival benefit. Emerging evidence indicates a role of sex hormones, genetics, and the immune system in modulation of both risk for TC and its progression in a sex-specific manner. Conclusion: Greater understanding of the molecular mechanisms underlying sex differences in TC pathogenesis could provide insights into the development of sex-specific, targeted, and effective strategies for prevention, diagnosis, and management. This review summarizes emerging evidence for the importance of sex in the pathogenesis, progression, and response to treatment in differentiated TC with emphasis on the role of sex hormones, genetics, and the immune system.

Comparing the rate and extent of malignancy in surgically excised thyroid nodules across race and ethnicity.

BACKGROUND: Few studies have compared the features of thyroid cancer among races and ethnicities. We hypothesized that race and ethnicity may influence the frequency and features of thyroid malignancy in thyroid nodules. METHOD: This was a retrospective chart review of patients between 2013 and 2020 who underwent thyroidectomy. RESULTS: In the analysis of 2737 patients, thyroid cancer was less prevalent among Blacks (24.0% vs Whites 52.1%, Hispanics 58.7%, Asians 71.7%, and Others 57.9%, p < 0.001). Thyroid cancer in Blacks was less likely to have extrathyroidal extension (9.7% vs Whites 18.6%, Hispanics 25.8%, Asians 18.2%, and Others 17.8%, p = 0.01), overall nodal involvement (12.4% vs Whites 31.1%, Hispanics 37.5%, Asians 36.3%, and Others 30.1%, p < 0.01), and lateral neck metastasis (4.4% vs Whites 10.8%, Hispanics 6.3%, Asians 13.2%, and Others 9.6%, p = 0.02). CONCLUSIONS: Race and ethnicity may play important roles in the risk of malignancy as well as in the extent of thyroid cancer.

Bone metastasis from noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP); a case report.

BACKGROUND: The term non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was recently proposed as a non-malignant thyroid lesion with indolent behavior that does not require post-operative radio-iodine treatment. We are reporting a case of NIFTP with bone metastasis that is the second case reported so far. CASE PRESENTATION: We describe a 38-year-old woman who presented with an indeterminate thyroid nodule and underwent total thyroidectomy with the finding of NIFTP on careful pathologic examination. However, her initial follow-up evaluation revealed a serum thyroglobulin level of > 300 ng/ml and a diagnostic whole body 131I scan demonstrated a focus of increased uptake in the left hemipelvis, confirmed on CT scan to be a lytic lesion in the left iliac bone. She was treated with 7.4GBq (200 mCi) of 131I and her follow-up 1 year later revealed an undetectable serum thyroglobulin and a negative whole body 131I scan with no visible uptake in the iliac bone indicating an excellent response. CONCLUSION: This case presentation reminds us to be alert to the rare occurrence of distant metastasis in NIFTP and the need for a case by case analysis and continuing post-operative follow-up for detection of residual or recurrent disease.

Biotin Interference in Assays for Thyroid Hormones, Thyrotropin and Thyroglobulin.

Background: Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This report describes a prospective study of possible biotin interference in thyroid-related laboratory assays, with a comparison of different commonly used assay platforms. Materials and Methods: Thirteen adult subjects (mean age 45 ± 13 years old) were administered biotin 10 mg/day for eight days. Blood specimens were collected at three time points on day 1 and on day 8 (baseline, two, and five hours after biotin ingestion). Thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroxine binding globulin (TBG), and thyroglobulin (Tg) levels were analyzed with four different platforms: Abbott Architect, Roche Cobas 6000, Siemens IMMULITE 2000, and liquid chromatography with tandem mass spectrometry (LC-MS/MS). TSH, fT3, fT4, TT3, and TT4 were measured with Abbott Architect and Roche Cobas 6000. fT3, fT4, TT3, and TT4 were also measured by LC-MS/MS. Tg was measured by Siemens IMMULITE 2000. TBG was assessed with Siemens IMMULITE 2000. Results: Significant changes in TSH, fT4, and TT3 measurements were observed after biotin exposure when the Roche Cobas 6000 platform was used. Biotin intake resulted in a falsely lower Tg level when measurements were performed with Siemens IMMULITE 2000. At the time points examined, maximal biotin interference was observed two hours after biotin exposure both on day 1 and day 8. Conclusions: A daily dose of 10 mg was shown to interfere with specific assays for TSH, fT4, TT3, and Tg. Physicians must be aware of the potential risk of erroneous test results in subjects taking biotin supplements. Altered test results for TSH and Tg can be particularly problematic in patients requiring careful titration of levothyroxine therapy such as those with thyroid cancer.

Do Molecular Profiles of Primary Versus Metastatic Radioiodine Refractory Differentiated Thyroid Cancer Differ?

Management of metastatic radioiodine refractory differentiated thyroid cancer (DTC) can be a therapeutic challenge. Generally, little is known about the paired molecular profile of the primary tumor and the metastases and whether they harbor the same genetic abnormalities. The present study compared the molecular profile of paired tumor specimens (primary tumor/metastatic sites) from patients with radioiodine refractory DTC in order to gain insight into a possible basis for resistance to radioiodine. Twelve patients with radioiodine refractory metastases were studied; median age at diagnosis of 61 years (range, 25-82). Nine patients had papillary TC (PTC), one had follicular TC (FTC), and two had Hürthle cell TC (HTC). Distant metastases were present in the lungs (n = 10), bones (n = 4), and liver (n = 1). The molecular profiling of paired tumors was performed with a panel of 592 genes for Next Generation Sequencing, RNA-sequencing, and immunohistochemistry. Digital microfluidic PCR was used to investigate TERT promoter mutations. The genetic landscape of all paired sites comprised BRAF, NRAS, HRAS, TP53, ATM, MUTYH, POLE, and NTRK genes, including BRAF and NTRK fusions. BRAF V600E was the most common point mutation in the paired specimens (5/12). TERT promoter mutation C228T was detected in one case. PD-L1 expression at metastatic sites was highly positive (95%) for one patient with HTC. All specimens were stable for microsatellite instability testing, and the tumor mutation burden was low to intermediate. Therefore, the molecular profile of DTC primary and metastatic lesions can show heterogeneity, which may help explain some altered responses to therapeutic intervention.

Optimal Time for 124I PET/CT Imaging in Metastatic Differentiated Thyroid Cancer.

BACKGROUND: The objective of this study was to determine the optimal time for 124I PET/CT imaging to maximize the detection of locoregional and/or distant metastases of differentiated thyroid cancer. METHODS: Differentiated thyroid cancer patients suspected of having metastatic disease were prepared with low-iodine diet and appropriate thyroid-stimulating hormone stimulation. 124I PET and low-dose localization CT were performed over 4 days after oral administration of 31.5 or 62.9 MBq (0.85 or 1.7 mCi) of 124I. Each scan was independently reviewed by 2 nuclear medicine physicians. All foci of activity were categorized, and the visual intensity of uptake was scored by a semiquantitative 3-point grading system (1: mild uptake, 2: moderate uptake, 3: intense uptake). Lesion volumes were determined on the CT image or on the PET images. Background (bkg) was also measured for each lesion and on each individual PET image. For each lesion, the mean activity concentration rate per unit administered activity (ACRmean/AA) and lesion-to-bkg ratios were compared across the 5 different time points. The semiquantitative grade and the quantitative measurements were compared. RESULTS: A total of 45 124I PET/CT scans were reviewed for 9 patients. In the visual assessment, a total of 31 foci suggestive for or highly suggestive of metastasis were identified on 124I PET/CT. Of these, 6 were seen on the 2-h, 18 on the 24-h, 27 on the 48-h, 24 on the 72-h, and 20 on the 96-h scan. There was a significant difference between the 24- and 48-h scans in the total number of foci (ie, locoregional and distant metastasis) (P < 0.05) and in the number of distant metastases (P < 0.05). The 24-, 48-, and 72-h scans identified the same number of locoregional foci. The 48-h scan visualized more of the distant metastases than any other time point. 124I PET/CT with dual-time-point imaging was superior to single-time-point imaging (97% vs 87%). In the quantitative analysis, the median ACRmean/AA was highest at 24 and 48 h, and the median lesion-to-bkg ratio was variable for different lesion locations. For lung metastases, the highest median lesion-to-bkg ratio was at 72 and 96 h. CONCLUSIONS: 124I PET/CT with dual-time-point imaging was superior to any single-time-point imaging (P < 0.10). Based on the visual assessment, dual time points at 48 + 72 h or 48 + 96 h yielded the highest lesion detection rate, whereas for single-time-point imaging, the 48-h images had the highest lesion detection rate. If the 48-h scan is completely negative or has negative 124I uptake in the region of interest, then a 72- or 96-h scan may be valuable. If lung metastases are suspected, then one should consider additional imaging at 72 or 96 h.