Causes of altered liver function tests - the role of alpha-1 antitrypsin.

BACKGROUND: Altered liver function tests are a common finding in clinical practice. Our retrospective study aimed to identify the diagnoses in a non-selected cohort of patients with altered liver tests and to investigate whether alpha-1 antitrypsin genotyping should be part of the diagnostic workup. PATIENTS AND METHODS: 501 patients who were admitted to our outpatient clinic for further evaluation of altered liver function tests were included in the study. The patients underwent a standardized diagnostic program with history taking, physical examination, laboratory tests and ultrasonography. Liver biopsy was performed if appropriate. RESULTS: More than 50 % of the patients had nonalcoholic fatty liver disease. Alcoholic and drug-induced liver injury were found in 8.6 % and 7 % of patients, respectively. Chronic hepatitis B and C, autoimmune liver disease and inherited causes of liver disease made up for approximately 16 % of the diagnoses. The remaining patients were diagnosed with kryptogenic liver disease or had miscellaneous diagnoses. In 3.7 % of the genotyped patients, the alpha-1 antitrypsin genotype PiMZ was found. CONCLUSION: Nonalcoholic fatty liver disease is nowadays the most frequent cause of altered liver tests. Alcoholic liver disease might be underrepresented in our study since these patients less often seek medical attention or the diagnosis is already made by the primary care physician. Drug-induced liver injury was found in more patients than expected and might therefore be underdiagnosed in practice. The alpha-1 antitrypsin genotype PiMZ was found in absence of other possible causes of liver disease, indicating that the PiMZ genotype is itself a risk factor for liver disease. Genotyping for alpha-1 antitrypsin should therefore be done when other causes for altered liver function tests have been ruled out.

Sedation-associated complications in endoscopy--prospective multicentre survey of 191142 patients.

BACKGROUND/AIMS: Propofol sedation is applied as moderate sedation for almost all diagnostic and interventional endoscopies. Propofol sedation bears the risk of complications such as respiratory as well as cardiopulmonary insufficiency including sedation-induced death. According to recent guidelines, non-anesthesiologist-administered propofol (NAAP) should be performed by an additional person who has NAAP as their sole task. METHODS: In a prospective multicentre survey involving 191,142 patients, clinically relevant endoscopy-associated complications were registered from 02/2010 to 01/2012. RESULTS: The majority of propofol sedations were applied without additional persons for NAAP. Overall endoscopy-related complication rate was 0.0022 % (n = 424) and sedation-related complications 0.00 042 % (n = 82). Variability over time and between the clinics was low and not influenced by the number of endoscopies performed during the investigation period. Sedation-related death occurred in 6 patients (0.00 003 %), 50 % during emergency endoscopies. In all sedation-associated deaths the patients had ASA class 3 before endoscopy. All fatal complications occurred in the presence of an additional trained person for NAAP. CONCLUSION: This large prospective survey shows that propofol sedation in gastrointestinal endoscopy is a safe procedure with a low potential of risk in daily routine. However, high risk patients (ASA ≥ 3) should be identified, especially before emergency endoscopies and managed by additional persons for NAAP and under intensive care surveillance.

Combination of bilateral metal stenting and trans-stent photodynamic therapy for palliative treatment of hilar cholangiocarcinoma.

Endoscopic biliary drainage is the mainstay of palliative treatment in patients with unresectable malignant hilar biliary obstruction. While self-expandable metal stents have shown significant advantages in distal tumors, bilateral hilar stenting is technically demanding. Moreover, ingrowth is a significant problem in uncovered stents. We evaluated the feasibility and efficacy of endoscopic bilateral JoStent SelfX deployment in patients with proximal malignant biliary obstruction in combination with photodynamic therapy (PDT) and/or chemotherapy. Twenty-one consecutive patients with malignant hilar biliary strictures were treated with transpapillary bilateral insertion of JoStentSelfX metal stents. Additional PDT was applied in 8 patients (PDT plus chemotherapy n = 4, only PDT n = 4). Solely chemotherapy was performed in 5 patients. Mean (+/- SD) stent patency was 173.9 +/- 201.8 days. The median estimated survival was 12.3 months (95 % CI: 8.5; 15.9). PDT was safely and efficaciously performed after endoscopic stent deployment (1.8 +/- 1.1 sessions/patient). There was a trend towards a longer stent patency in patients receiving additional therapy (202.2 +/- 197.6 vs. 128 vs. 213.2 days; p = 0.38). Furthermore, we observed a significantly longer survival in this cohort (16.5 [12.2; 20.1] vs. 12.3 [1.9; 8.5] months, p < 0.005). Additional therapy had no significant impact on cumulative hospitalization time (16.3 +/- 15.8 vs. 14.4 +/- 22.5 days; p = 0.54). Bilateral insertion of Jostent SelfX in patients with proximal cholangiocarcinoma is feasible and effective and can be safely combined with trans-stent photodynamic therapy.

Short-term effects of transjugular intrahepatic shunt on cardiac function assessed by cardiac MRI: preliminary results.

The purpose of this study was to assess shortterm effects of transjugular intrahepatic shunt (TIPS) on cardiac function with cardiac magnetic resonance imaging (MRI) in patients with liver cirrhosis. Eleven patients (six males and five females) with intractable esophageal varices or refractory ascites were imaged with MRI at 1.5 T prior to, within 24 h after, and 4-6 months after TIPS creation (n = 5). Invasive pressures were registered during TIPS creation. MRI consisted of a stack of contiguous slices as well as phase contrast images at all four valve planes and perpendicular to the portal vein. Imaging data were analyzed through time-volume curves and first derivatives. The portoatrial pressure gradient decreased from 19.8 + or = 2.3 to 6.6 + or = 2.3, accompanied by a nearly two fold increase in central pressures and pulmonary capillary wedge pressure immediately after TIPS creation. Left and right end diastolic volumes and stroke volumes increased by 11, 13, and 24%, respectively (p\0.001), but dropped back to baseline at follow-up. End systolic volumes remained unchanged. E/A ratios remained within normal range. During follow-up the left ventricular mass was larger than baseline values in all patients, with an average increase of 7.9 g (p\0.001). In conclusion, the increased volume load shunted to the heart after TIPS creation transiently exceeded the preload reserve of the right and left ventricle, leading to significantly increased pulmonary wedge pressures and persistent enlargement of the left and right atria. Normalization of cardiac dimensions was observed after months together with mild left ventricular hypertrophy.

Biliary metal stents and air embolism: a note of caution.

Metal stents are a valuable treatment modality for patients with biliary obstruction. However, we present here two patients whose cases may serve as a warning about an unusual complication associated with these stents. We encountered this complication after endoscopic retrograde cholangiography for obstructed metal biliary stents. The first patient, an 87-year-old man with a benign biliary stricture, failed to regain consciousness after clearing of his stent using a Dormia basket and balloon catheter. Cerebral air embolism was diagnosed on cerebral computed tomography, and transesophageal echocardiography revealed a patent foramen ovale as a precipitating factor for paradoxical air embolism. He survived and was discharged with a residual hemiparesis. In the second patient, a 54-year-old man who had a history of a Billroth II operation and chronic pancreatitis and who had a portal cavernoma with biliary obstruction due to collateral veins, electromechanical dissociation complicated the balloon-catheter stent revision. Echocardiography performed during cardiopulmonary resuscitation showed major air embolism to the right heart. The patient died. These cases demonstrate that air may gain access to the venous system during therapeutic endoscopic procedures of this type. It is likely that the large diameter of metal stents and the potential for these stents to lacerate venous structures facilitate the entry of air into the venous circulation, an event which may have life-threatening consequences.

A patient with portal hypertension and blindness after transjugular intrahepatic portosystemic shunt.

We report on a case of recurrent variceal bleeding from gastric varices, which was treated with transjugular intrahepatic portosystemic shunt (TIPS) and Histoacryl injection into the gastric varices. Furthermore, the patient had a small patent foramen ovale without a right-to-left shunt. After the intervention, the patient developed acute neurological disorders as a result of a cerebral paradoxical embolism. In the following, we describe the potential risk of histoacryl in paradoxical embolization when used for the injection of variceal collaterals during TIPS placement in patients with portal hypertension. The present case report shows a very rare but important complication after TIPS implantation. To avoid this complication it is recommended to perform echocardiography before all TIPS placements.

Magnetic resonance (MR) imaging and MR angiography for evaluation and follow-up of hepatic artery banding in patients with hepatic involvement of hereditary hemorrhagic telangiectasia.

BACKGROUND: We describe findings obtained by magnetic resonance angiography (MRA) and magnetic resonance imaging (MRI) for evaluation and follow-up after hepatic artery banding in patients with hepatic involvement of hereditary hemorrhagic telangiectasia (HHT). METHODS: Abdominal MRA and liver MRI were performed in three patients with HHT as clinically defined by Curacao criteria. One patient underwent MRA and MRI twice for preinterventional evaluation and follow-up, one patient for preinterventional evaluation, and one patient for postinterventional evaluation. Hepatic vascular involvement of the disease and postinterventional vascular anatomy were evaluated by two radiologists by consensus. RESULTS: Hepatic vascular involvement with perfusion disorders and arteriosystemic shunts was found in all three patients. MRA and MRI allowed diagnostic characterization of hepatic vascular disease (three of three), preinterventional evaluation of complex vascular anatomy and variants (two of two), and postinterventional follow-up of hepatic artery banding (two of two). CONCLUSION: In preinterventional evaluation and postinterventional follow-up, MRA and MRI allows characterization of complex hepatic vascular alterations of HHT and, hence, is an alternative to other imaging modalities in the diagnosis, clinical decision making, and follow-up of HHT.

[Gastrointestinal bleeding in portal hypertension in liver cirrhosis]. / Die intestinale Blutung bei portaler Hypertension bei Leberzirrhose.

There are three major goals in the prophylaxis and treatment of upper gastrointestinal bleeding in portal hypertensive patients: prophylaxis of the first bleeding episode, therapy of active bleeding and prophylaxis of recurrent bleeding. Several therapeutic options are available: non-selective beta-blockers are the treatment of choice in the primary prophylaxis of the first bleeding episode in patients with large esophageal varices. Alternatively, endoscopic band ligation therapy is an option. Acute bleeding varices should be treated by ligation pharmacological and antibiotic therapy. Prophylaxis of recurrent bleeding is patient-dependent: shunt surgery is an option in young patients in a good medical condition (Child-Pugh class A). In patients with refractory ascites and a bilirubin below 3 mg/dl, TIPS is a good option together with recurrent bleeding. At the moment, there are no trials showing that endoscopic ligation therapy is superior to prevent pharmacological therapy. Nevertheless, the first-line treatment in most patients in Germany is endoscopic band ligation. Bleeding from ectopic varices and bleeding from hypertensive gastropathy should be treated individually either by endoscopy, TIPS or drug therapy.

Acute haemodynamic effects of losartan in anaesthetized cirrhotic rats.

BACKGROUND: Portal hypertension in cirrhosis is the result of increased intrahepatic vascular resistance to portal outflow as well as increased portal tributary blood flow. The angiotensin II type 1 receptor antagonist losartan has been suggested as a portal pressure-lowering drug in patients with cirrhosis. AIM: To investigate the systemic and splanchnic haemodynamic effects of different doses of losartan. METHODS: In 35 anaesthetized rats with secondary biliary cirrhosis, 3, 10 or 30 mg of losartan kg(-1) or solvent were administered intravenously. Ten sham-operated rats served as controls. Mean arterial pressure and portal pressure were measured by catheters in the femoral artery or portal vein. Systemic and splanchnic haemodynamics and mesenterico-systemic shunt rate were determined by the coloured microsphere method. RESULTS: Losartan reduced portal pressure (sham: 9.1 +/- 0.4. cirrhosis: 19.3 +/- 1.1, after 3 mg kg(-1) of losartan 16.4 +/- 0.4, after 10 mg kg(-1) of losartan 15.6 +/- 0.6, after 30 mg kg(-1) of losartan 14.9 +/- 0.6 mmHg) without reducing portal sinusoidal resistance. However, in cirrhotic rats it reduced portal tributary blood flow (sham: 4.3 +/- 0.6. cirrhosis: 8.6 +/- 1.4, after 3 mg kg(-1) of losartan 3.8 +/- 0.7, after 10 mg kg(-1) of losartan 4.7 +/- 0.5, after 30 mg kg(-1) of losartan 5.9 +/- 0.9 mmHg). This was owing either to an increase in splanchnic vascular resistance at the 3 mg kg(-1) dose or to a reduction in the splanchnic perfusion-pressure gradient secondary to a reduction in mean arterial pressure at the 10 and 30 mg kg(-1) doses (mean arterial pressure: sham: 109.7 +/- 4.8. cirrhosis: 109.4 +/- 2.8, after 3 mg kg(-1) of losartan 99.7 +/- 2.9, after 10 mg kg(-1) of losartan 89.9 +/- 3.4, after 30 mg kg(-1) of losartan 81.0 +/- 2.9 mmHg). CONCLUSIONS: Low doses of losartan reduce portal hypertension by an increase in splanchnic vascular resistance without hypotensive side-effects on arterial pressure.

[Chronic diarrhea in a 43-year-old patient]. / Chronische Diarrhö bei einem 43-jährigen Patienten.

A 43-year old patient came to our clinic with chronic diarrhea. Determination of the faecal alpha 1-antitrypsin-clearance led to the diagnosis of exsudative enteropathy. Blood counts showed pronounced lymphocytopenia. Histopathological findings from intestinal and colorectal biopsies showed diffuse lymphangiectasis. Following exclusion of secondary types, our diagnosis was primary intestinal lymphangiectasis. Additional distinctive morphological and anamnestic features strongly suggested presence of Noonan's syndrome. Characteristic manifestations of Noonan's syndrome include changes in the lymphatic vessels in accordance with primary lymphangiectasis. Frequently, these changes are localized in the lungs. To date, only rare cases of intestinal lymphangiectasia in Noonan's syndrome have been reported. Treatment consisted of a protein-rich diet, with reduced fat content enriched by middle-chain fatty acids, as well as twice-daily injections of 200 micrograms octreotide. With this therapy, the symptoms improved.

Portal hypertension is associated with increased mRNA levels of vasopressor G-protein-coupled receptors in human hepatic arteries.

BACKGROUND: The contractile response of human splanchnic vessels to different vasoconstrictors is attenuated in cirrhosis. Functional studies indicate a cellular signalling defect upstream of the G-protein level. The aim of the present study was to analyze expression and mRNA levels of the following most relevant vasopressor receptors in the smooth musculature of human hepatic arteries: alpha1 adrenoceptor (AR) subtypes a, b and d, angiotensin II type 1 receptor (AT1), arginine vasopressin receptor type 1a (V1a), endothelin receptor type A (ETA) and B (ETB). MATERIALS AND METHODS: Hepatic arteries were collected from 10 donors (noncirrhotic) and 14 recipients (cirrhotic) at liver transplantations. Real-time-PCR was performed to quantify steady-state levels of receptor mRNAs. RESULTS: alpha 1aAR mRNA levels showed no significant difference between the cirrhotic arteries and the controls while the mRNA levels of the other vasoactive receptors were significantly higher in the cirrhotic hepatic arteries (alpha 1bAR: 4-fold, P = 0.013; AT1: 16-fold, P = 0.024; V1a: 23-fold, P = 0.001; ETA: 4-fold, P = 0.02; ETB: 8-fold, P = 0.008). No mRNA for the alpha 1dAR was detected either in the donor or recipient hepatic arteries. CONCLUSION: We conclude that vascular hyporeactivity to the most relevant endogenous vasoconstrictors of cirrhotic hepatic arteries is not caused by a receptor down-regulation at mRNA levels. In contrast they were up-regulated.

[MRI in cavernous transformation of the portal vein: secondary biliary abnormalities and portoportal collaterals]. / MRT bei kavernöser Transformation der Pfortader - Sekundäre Gallengangsveränderungen und portoportale Kollateralen.

PURPOSE: Evaluation of portoportal collateral vessels and associated biliary abnormalities in patients with cavernous transformation of the portal vein by MRI. MATERIAL AND METHODS: Review of 34 MRI examinations performed on patients with angiographically or MR-angiographically proven cavernous transformation. The analysis included the pattern of the portoportal collateral circulation and the abnormalities of the biliary system, such as wall thickening, stenosis, dilations and irregularities of the extra-and intrahepatic bile ducts. RESULTS: 23 (67.6%) of 34 patients with cavernous transformation had paracholedochal portoportal collateral vessels, with 22 (64.7%) showing visible luminal channels. Epicholedochal venous collaterals could be observed in 8 (23.5%) patients. 24 (70.5%) of 34 patients demonstrated biliary abnormalities due to portoportal collaterals, leading to stenosis with dilatation of the proximal bile ducts in 8 (23.5%) patients. The ductal walls were irregular in 7 (20.5%) patients, and thickened in 11 (32.3%). The gallbladder wall was thickened in 4 (12.9%) patients. CONCLUSION: Portoportal collaterals in patients with cavernous transformation of the portal vein can be identified by MRI. These collaterals frequently alter the biliary system, which must be considered in differential diagnosis of biliary abnormalities observed in the presence of portoportal collaterals.

Hemodynamic effects of propranolol and nitrates in cirrhotics with transjugular intrahepatic portosystemic stent-shunt.

BACKGROUND: The combination of tailored TIPS with vasoactive drugs might allow reduction of the rate of subsequent shunt-related sequelae. METHODS: We studied cirrhotic patients 8 weeks (median) after TIPS insertion (8-10 mm) for variceal bleeding. Nitrate (0.1 mg/kg) and propranolol (0.15 mg/kg) alone or combined (same dosages) were infused (I h) sequentially at 1-h intervals (n = 17). Similarly, propranolol was randomly compared to placebo (NaCl, n = 14). We measured mean arterial pressure (MAP, mmHg), heart rate (HR) and portal pressure gradient (PPG: portal minus central venous pressure) prior to and after drugs. RESULTS: Propranolol reduced PPG (mean +/- s, mmHg) significantly (14.8 +/- 3.7 versus 12.1 +/- 3.7; -21% +/- 10%; P < 0.001), while nitrates alone (14.3 +/- 3.4 versus 13.7 +/- 3.4; -11% +/- 3%; P=0.06) or nitrates plus propranolol (12.9 +/- 4 versus 12.4 +/- 4; -7% +/- 8%; P=0.2) induced only minor additive effects on portal pressure. However, nitrate reduced MAP (P < 0.001) and increased HR (P < 0.01), whereas propranolol reduced only HR (P < 0.001) with unchanged MAP, and the combination decreased MAP (P < 0.001). Compared to placebo (no effect), propranolol decreased PPG (14.4 +/- 5.6 versus 11.1 +/- 5.5; -23% +/- 11%; P < 0.001) and HR (P < 0.001). Overall, most patients (92%) responded to propranolol and 54% showed a marked PPG decrease (>20%). CONCLUSIONS: Propranolol significantly reduced portal pressure in cirrhotic patients after TIPS, whereas nitrates induced only minor benefit. TIPS-treated patients might therefore profit from additive propranolol therapy allowing limited shunts to be applied initially and/or to reduce the need for TIPS revisions in the case of shunt-dysfunction during follow-up.

Long-term results and prognostic factors in the treatment of achalasia with botulinum toxin.

BACKGROUND AND STUDY AIMS: Endoscopic therapy of achalasia by injection of botulinum toxin into the lower esophageal sphincter has very limited adverse effects and is initially successful in 70 % of patients. However, this result only lasts for 6 - 9 months on average in most patients and only half of them benefit for more than 1 year. The aim of this study was to find out which factors are predictive for a good long-term success. PATIENTS AND METHODS: We retrospectively studied 25 patients with achalasia. The diagnosis had been proven by barium swallow and esophageal manometry. Therapy was carried out endoscopically between June 1996 and December 1998 by injection of 25 mouse units (MU) botulinum toxin into each of the four quadrants of the lower esophageal sphincter (LES). Lower esophageal sphincter pressure (LESP) was measured prior to and 1 week after endoscopic therapy. A standardized questionnaire was used for symptom assessment, at the initial presentation, at 1 week and at 2.5 +/- 0.8 years after treatment. RESULTS: The LESP was significantly reduced (pre-treatment 62.1 +/- 15.2 mmHg vs. post-treatment 43.1 +/- 12.5 mmHg; P < 0.01). Symptoms improved in 16 patients (pre-treatment symptom score 9.5 +/- 2.9 vs. post-treatment symptom score 4.7 +/- 1.8; P < 0.01). Nine patients showed no relevant improvement. From the 16 patients with a good initial response, two were lost to follow-up. In nine patients the outcome was still satisfactory after a mean of 2.5 years (1.5 - 4 years) (pre-treatment symptom score 9.5 +/- 2.9 vs. symptom score at 2.5 years after Botox 5.1 +/- 1.5; P < 0.05). These patients were on average 67.7 +/- 12.5 years old. The remaining five patients received a second or third injection of botulinum toxin, but none improved substantially for more than 6 months. One of them eventually underwent pneumatic dilation, and three laparoscopic myotomy. Thus, botulinum toxin treatment was unsuccessful in 14 patients in all. These 14 patients were, on average, significantly younger than the nine successfully treated patients (46.1 +/- 12.6 years vs. 67.7 +/- 12.5 years; P < 0.01) and had significantly higher LESP values prior to botulinum toxin therapy (72.8 +/- 8.9 mmHg vs. 47.8 +/- 9.2 mmHg; P < 0.01). CONCLUSIONS: The long-term success of botulinum toxin injection into the LES in patients with achalasia is highest in elderly patients and in patients with an LESP not exceeding the upper normal level prior to treatment by 50 % or more. On the basis of our results, younger patients (< 55 years) with a severe increase in LESP do not seem to benefit from botulinum toxin injection and pneumatic dilation or myotomy may be more advantageous to them.

Transjugular intrahepatic portosystemic shunting is not superior to endoscopic variceal band ligation for prevention of variceal rebleeding in cirrhotic patients: a randomized, controlled trial.

BACKGROUND: The aim of the present study was to compare the transjugular intrahepatic portosystemic shunt (TIPS) with variceal band ligation (VBL) in the prophylaxis of variceal rebleeding in patients with cirrhosis of the liver. METHODS: Fifty-four cirrhotic patients (21 Child-Pugh class A, 27 class B, 6 class C) were randomized to TIPS (n = 28) or VBL (n = 26) within 2 months after control of esophageal variceal hemorrhage. Statistical analysis was performed on the intention-to-treat principle. RESULTS: Mean follow-up was 2 years. Mortality risk at 1 and 2 years of follow-up was 7.8% +/- 5.3% and 19.9% +/- 8.8% in the TIPS group and 16.5% +/- 7.6% and 16.5% +/- 7.6% in the VBL group, respectively (n.s.); actuarial probability of remaining free from rebleeding was 83.7% +/- 77.4% and 71.4% +/- 10.4% in the TIPS group and 83.9% +/- 7.3% and 78.1% +/- 8.8% in the VBL group at 1 and 2 years, respectively (n.s.). Hepatic encephalopathy within 1 month after randomization was observed in 2 patients in the TIPS group and in 1 in the VBL group. CONCLUSION: TIPS is not superior to VBL in the prevention of variceal rebleeding. Furthermore, similar mortality rates in patients treated with TIPS or VBL negate TIPS as the preferred strategy for prevention of variceal rebleeding.

Contractile hyporesponsiveness of hepatic arteries in humans with cirrhosis: evidence for a receptor-specific mechanism.

Splanchnic vasodilatation and vascular hyporesponsiveness to vasopressors are characteristic features of patients with cirrhosis. Although the vascular response to different vasopressors has been shown to be attenuated in cirrhosis, alterations on the receptor level are discussed controversially. Thus, impaired postreceptor signaling has been postulated. However, so far this has not been studied in human splanchnic vessels. Therefore, we assessed the vascular response of human hepatic arteries after activating the G-protein-dependent signal transduction pathway by stimulation with angiotensin II, the thromboxane A(2) analog U46619, or by G-protein activation with NaF/AlCl(3). After endothelium denudation, cumulative isometric concentration contraction curves were obtained for hepatic arteries from 32 cirrhotic patients undergoing liver transplantation and from 40 organ donors after stimulation with either angiotensin II (10(-11)-10(-5) mol/L), U46619 (10(-10)-10(-6) mol/L) or AlCl(3) (30 micromol/L)/NaF (10(-4)-3 x 10(-2) mol/L). Hepatic arteries from cirrhotic patients were markedly less responsive to angiotensin II (P <.0001) than those from organ donors. Both stimulation of the G-protein phospholipase C pathway via the thromboxane A(2) receptor and receptor-independent G-protein stimulation with AlCl(3)/NaF, induced an intact contractile response. In conclusion, the G-protein-dependent signal transduction system itself is unaltered in cirrhosis. Hence, the cause of the hyporesponsiveness to some vasoconstrictors in cirrhosis appears to be a receptor-specific phenomenon localized upstream from the G-protein level.