Whales, lifespan, phospholipids, and cataracts[S]

This study addresses the question: why do rats get cataracts at 2 years, dogs at 8 years, and whales do not develop cataracts for 200 years? Whale lens lipid phase transitions were compared with the phase transitions of other species that were recalculated. The major phospholipids of the whale lens were sphingolipids, mostly dihydrosphingomyelins with an average molar cholesterol/phospholipid ratio of 10. There was a linear correlation between the percentage of lens sphingolipid and lens lipid hydrocarbon chain order until about 60% sphingolipid. The percentage of lens sphingolipid correlated with the lens lipid phase transition temperature. The lifespan of the bowhead whale was the longest of the species measured and the percentage of whale lens sphingolipid fit well in the correlation between the percentage of lens sphingolipid and lifespan for many species. In conclusion, bowhead whale lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing these lenses to stay clear relatively longer than many other species. The strong correlation between sphingolipid and lifespan may form a basis for future studies, which are needed because correlations do not infer cause. One could hope that if human lenses could be made to have a lipid composition similar to whales, like the bowhead, humans would not develop age-related cataracts for over 100 years.

Lipid oxidation is obviously deleterious to the lens and, based on inter-species differences in lens phospholipid composition, it has been suggested that humans have adapted so that their lens membranes have a high sphingolipid (lower amount of double bonds) content that confers resistance to oxidation, allowing their lenses to stay clear for a longer time relative to those in many other species (35). However, humans were the only species tested that have a lifespan of over 70 years, so to further test the idea above, in this study we measured membrane lipid structure and the phospholipid composition of bowhead whale lenses. Bowhead whales (Balaena mysticetus) may be the oldest mammals that exist with lifespans of over 200 years (49)(50)(51)(52)(53)(54) and do not get cataracts (55). Differences in animal lens lipid composition could explain why lanosterol ameliorates cataracts in dogs and rabbits (56), but is ineffective in reversing cataracts in humans (57).

Sample collection
For several decades, scientists with the North Slope Borough Department of Wildlife Management have worked closely with Eskimo whaling captains from the 11 Alaskan whaling communities to examine bowhead whales taken during subsistence hunts (58). Aboriginal bowhead whale hunting occurring during spring and fall in 11 Alaskan whaling communities has been regulated by a quota system under the authority of the International Whaling Commission since 1977. During this longstanding cooperative research relationship, a large number of eyes from freshly dead whales have been preserved (frozen intact at 20°C) along with other tissue samples and basic biological data for each whale. Marine mammal tissue samples used in this study were collected under National Marine Fisheries Service permits 17350-00 and 17350-01 issued to the North Slope Borough Department of Wildlife Management.

Globe enucleation, lens dissection, and lipid extraction
Unlike globe enucleation from humans and animals obtained from the local abattoir, whose death and globe dissection occur at room temperature, whales globes were harvested in ideal conditions. The ambient air and water temperature at the time of the whale harvest was at or below freezing. From time of death to sample collection (i.e., whales landed) it took on average 5-6 h. A scientist was present to remove the globe immediately upon landing and the globe was one of the first of the tissues collected. Previously frozen whale lenses were removed from the globe by an anterior approach. The iris and ciliary body were carefully removed from the lens by cutting the zonules with curved micro scissors.
Lenses were each extracted using a monophasic methanol extraction procedure that gave the highest yield of lens phospholipid compared with other extraction protocols (59). Lenses were each added to 5 ml of methanol (Sigma-Aldrich, St. Louis, MO) in a 25 ml glass scintillation vial (Fisher Scientific, Pittsburgh, PA) and chopped using a Teflon™ coated spatula. Argon gas (analyzed, ultra-pure; Welders Supply, Louisville, KY) was bubbled into the methanol/lens mixture for 2 min to remove oxygen. The vial was sonicated in an ultrasonic bath (model 1510; Branson Ultrasonics, Danbury, CT) for 10 min. The mixture was centrifuged for 10 min at 10,000 g. The supernatant was decanted and dried with a stream of argon gas. Methanol (5 ml) was added to the pellet and the procedure above, from the bubbling of argon gas on, was repeated two times with the supernatant from the centrifugation step added to the supernatant(s) from the previous steps and dried with a stream of argon gas. To ensure all methanol was removed from the sample, it was dried further in a lyophilizer set at 29 torr and a condenser temperature <50°C (Labconco, Kansas City, MO; Freeze Dryer 3) for 1 h. CDCl 3 (1 ml; Sigma-Aldrich) was added to the dried lipid. The vial was sonicated in an ultrasonic bath (model 1510; Branson Ultrasonics, Danbury, CT) for 10 min and the mixture was placed into a 9 mm microvial with a Teflon™ cap (Microliter Analytical Supplies, Suwanee, GA) to which argon gas was added. Samples were stored for no more than 24 h at 20°C.
Cholesterol to phospholipid ratio analysis using 1

H-NMR
Half (500 l) of the lens lipid-CDCl 3 extract was transferred from the microvial to an NMR tube using a glass pipette. Spectral data were acquired using a Varian VNMRS 700 MHz NMR spectrometer (Varian, Lexington, MA) equipped with a 5 mm 1 H{ 13 C/ 15 N} 13 C enhanced PFG cold probe (Palo Alto, CA). Spectra were acquired with a minimum of 250 scans, 45° pulse width, and a relaxation delay of 1.0 s. All spectra were obtained at 25°C. Spectra were processed and integration of spectral bands was performed with GRAMS/386 software (Galactic Industries, Salem, NH). Cholesterol to phospholipid molar ratios were calculated as discussed in the Results. 31

P-NMR
A methanol/0.2 M Cs-EDTA (4:1) reagent (60) was added (250 l) to the sample used for 1 H-NMR spectroscopy and homogenized with the use of a temperature-controlled ultrasonic bath at 40°C for 10 min. 31 P-NMR spectra were measured using a Varian Inova-400 spectrometer. The parameters to perform the acquisitions were similar to those described previously (61). Briefly, a spectral width of 2,024.7 Hz (sweep width  = 10 ppm), 60° pulse, 4,000 data points, 1.0 s delay time, 0.711 s acquisition time, and proton decoupling (WALTZ) at 500.16 MHz were used. A line broadening of 3.0 Hz and phase correction were used to process the spectra. Chemical shifts were referenced to internal or added bovine brain sphingomyelin (SM) (Sigma-Aldrich) resonance at  = 0.09 ppm. Spectra were processed and integration of spectral bands was performed with GRAMS/386 software (Galactic Industries, Salem, NH).

Measurement of lipid phase transitions using Fourier transform infrared spectroscopy
The 500 l of sample not used for NMR analysis was applied to an AgCl infrared window. The solvent was evaporated under a stream of argon gas and the window was placed in a lyophilizer for 4 h to remove all traces of solvent. Infrared spectra were measured using a Fourier transform infrared spectrometer (Nicolet 5000 Magna Series; Thermo Fisher Scientific, Inc., Waltham, MA). Lipid on the AgCl window was placed in a temperature-controlled infrared cell. The cell was jacketed by an insulated water coil connected to a circulating water bath (model R-134A; Neslab Instruments, Newton, NH). The sample temperature was measured and controlled by a thermistor touching the sample cell window. The water bath unit was programmed to measure the temperature at the thermistor and to adjust the bath temperature so that the sample temperature could be set to the desired value. The rate of heating or cooling (1°C/15 min) at the sample was also adjusted by the water bath unit. Temperatures were maintained within ±0.01°C. Exactly 100 interferograms were recorded and averaged. Spectral resolution was set to 1.0 cm 1 . Infrared data analysis was then performed (GRAMS/386 software; Galactic Industries, Salem, NH). The frequency of the symmetric CH 2 stretching band near 2,850 cm 1 (ṽ sym ) was used to estimate the content of trans and gauche rotamers in the hydrocarbon chains. The ṽ sym was calculated by first baseline leveling the OH-CH stretching region between 3,500 and 2,700 cm 1 . The center of mass of the CH symmetric stretching band was calculated by integrating the top 10% of the intensity of the band. The baseline for integrating the top 10% of the intensity of the band was parallel to the OH-CH region baseline. The change in ṽ sym versus temperature was used to characterize lipid phase transitions, as described previously (62). Because rotamers are either in trans or gauche conformations, phase transitions were fit to a two-state sigmoidal equation using Sigma plot 10 software (Systat Software, Inc., Chicago, IL): The ṽ sym is the frequency of the symmetric CH 2 stretching band near 2,850 cm

1
. Tc is the phase transition temperature. Hillslope is a measure of the degree to which one lipid influences the melting of adjacent lipids. The steeper the phase transition, the larger is the hillslope.
Lipid order at 25 and 33.4°C was calculated by extrapolating the ṽ sym at 25 and 33.4°C from the fit of the phase transition and then converting ṽ sym to the percentage of trans rotamers, a measure of lipid conformational order (62). The data for percentage of trans rotamer were used to calculate the phase-transition change in enthalpy (H) and entropy (S) from the slopes of Arrhenius plots (62).

Estimation of whale age
Three published methods for estimating the age of bowhead whales were used in this analysis (51,52,54). For this work, a mean and SE was not calculated, rather an age range was determined for each of the three animals and the pool of whales.

Statistics
Curves were fit using Sigma plot 10 software (Systat Software, Inc.) and the confidence levels, P, were obtained from a critical value table of the Pearson product-moment correlation coefficient. A value of P < 0.01 was considered statistically significant.

RESULTS
Bowhead whale eyes were collected from seven female and six male legally subsistence-harvested bowhead whales in 2011, 2013, 2014, and 2015. Total body length measured from the rostrum to the fluke notch ranged between 8.2 and 17.2 m. The length of four of the animals was greater than 13 m, thus they were mature (51). The demographic of the samples are summarized in Table 1. NMR spectroscopy 31 P-NMR was used to quantify whale lens phospholipids (Fig. 1). The major phospholipids of the bowhead whale lens were sphingolipids, which composed 60% to 100% of the total phospholipid (Table 1). For two of the lenses, only one 31 P-NMR resonance for DHSM was visible. To confirm that the resonance assignment was correct, the sample was spiked with bovine SM and, indeed, the resonances for SM (0.090 ppm) and DHSM (0.117 ppm) were in the correct position ( Fig. 1iii) (63). The lenses from the two largest (13B1; 14B6) and thus oldest whales weighed the most and had the most DHSM (Table 1).
Cholesterol was quantitated from the 1 H-NMR spectra of the lens lipid extracts. Phospholipid was quantified from the amide NH resonances near 6.81 and 6.22 ppm (Fig.  2B) assigned to hydrated and partially hydrated sphingolipid, respectively (63,64), and the resonance near 3.48 ppm ( Fig. 2A) assigned to the head group N-CH 2 moiety (63)(64)(65). The assignments were reasonable as the resonances were in the correct position and the measured average NCH 2 /amide-NH intensity ratio was 1.9, close to the expected intensity ratio of 2. Cholesterol was quantified from the CH 3 resonance near 0.67 ppm (Fig. 2C) assigned to carbon 18 and the CH resonance near 1.83 ppm (Fig.  2D) assigned to H1, H2, and H16 of the cholesterol molecule (66)(67)(68). A proton resonance from the cholesterol ester at 4.6 ppm was not detected, indicating that most or all of the cholesterol was not esterified. The assignments are reasonable, as the resonances were in the correct position and the measured (3  CH)/CH 3 average intensity ratio was 0.96, close to the expected intensity ratio of 1. The four combinations of cholesterol/phospholipid intensity ratios had a relative SE of 4% for each sample (Table 1). The average molar cholesterol/phospholipid ratio for all four samples was similar, 10 ± 0.7 mol cholesterol per mole phospholipid (Table 1).

Infrared spectroscopy
Infrared spectroscopy was used to measure lipid-lipid interactions and the composition of whale lipid suspensions. The CH 2 stretching and bending bands were predominant in the infrared spectra of lipids due to the large number of CH 2 groups in their hydrocarbon chains (Fig. 3). Tentative infrared band assignments are listed in Table 2. Note that the intensity of the carbonyl band near 1,740 cm 1 is very small compared with the intensity of the amide band near 1,650 cm 1 and the CH 2 bending band near 1,470 cm 1 , which supports the large percentage of ether and amide linked hydrocarbon chains measured by 31 P-NMR (Fig.  3B). Note also the similarities between the spectrum of the whale lens lipid extract (Fig. 3Bi) and that of bovine brain SM (Fig. 3Bii) and cholesterol (Fig. 3Biii).
The CH stretching region near 2,900 cm 1 , is composed of six major bands (Fig. 3A) (62,69). In this study, we used the ṽ sym near 2,850 cm 1 to estimate the trans to gauche rotamer content of the hydrocarbon chains (Fig. 3A). The ṽ sym increased with an increase in temperature and the number of gauche rotamers, concurrent with a decrease in intensity (Fig. 4) (69). Lipid phase transitions were quantified by fitting them to a four-parameter two-state sigmoidal equation, as described by Borchman et al. (62). The four parameters fitted were the minimum and maximum ṽ sym in the phase transition that corresponded to the most ordered and disordered states, respectively, the transition temperature at which half of the lipid molecules underwent a phase change, and the relative cooperativity. The broader the phase transition, the smaller the absolute value of the cooperativity. Cooperativity describes how the order of a lipid influences that of neighboring lipids. The four phase-transition parameters necessary for defining the phase transition and other parameters calculated from the defining parameters are listed in Table 3.
Lipid order was measured close to the temperature of the lens, 33.4°C, by extrapolating the ṽ sym at 33.4°C from the fit of the phase transition and then converting ṽ sym to the percentage of trans rotamers (Table 3) (61). Lipid order at 25°C was also calculated (Table 3) because the temperature of whale lenses exposed to extremely low arctic temperatures could be lower than that for humans. The phase transition was relatively broad, which indicates that the cooperativity between lipids was low and changes in lipid order with temperature were minimal.

Recalculating phase transition parameters from previous studies
We have measured lens lipid phase transitions for lipids extracted from a number of species, including rabbit (70),

31
P-NMR spectra of phospholipids extracted from bowhead whale lenses. Spectra were used to quantify the phospholipid composition in Table 1. Pool (i), whale C (ii), whale A plus SM standard (iii), whale A (iv), Whale B (v). DHSM, dihydrosphpningomyelin; PEe, phosphatidylethanolamine ether; PSe, phosphatidylserine ether; PCe, PC ether. bovine (71), rat and camel (35), guinea pig (72), and humans (8,73,74). We refit the phase transition curves from previous studies and recalculated the percent trans rotamers because the four phase transition parameters used to define the whale lens lipid transition in the current study were not reported for other species. Also, in previous publications (35,(70)(71)(72)(73)(74), the equation used to curve fit the phase transitions was a general equation for sigmoidal curves. The equation 1 used in the current study is more physiologically relevant, as it is related to the "Hill" equation used to measure enzyme kinetics. Another reason to recalculate the previously measured phase transitions is the minimum and maximum ṽ sym used in the older studies were less accurate. In previous studies (8,35,(70)(71)(72)(73)(74), the maximum ṽ sym of 2,854.5 cm 1 was estimated from phosphatidylcholine (PC) in CHCl 3 . In this study, we used a maximum ṽ sym of 2,855.36 cm 1 calculated from an isomeric distribution of hexanes (62). Also, in previous studies (8,35,(70)(71)(72)(73)(74), the minimum ṽ sym of 2,849 cm 1 was estimated from dipalmitoylphosphatidylcholine at 20°C. In this study, we used a minimum ṽ sym of 2,848.00 cm 1 calculated from distearoylphosphatidylcholine at 50°C (62). The recalculated phase transition parameters are listed in Table 4 for comparison with the phase transition parameters for whale lenses calculated in this study. Arrhenius plots used to calculate the H and S values from the lipid phase transitions were linear, with correlation coefficients greater than 0.998.

Lens growth
The average lens growth of the bowhead whale was 4.4 mg/year calculated from the slope of the linear relationship (r = 0.539, n = 13, P < 0.05) between lens weight and body length (0.0505 g/m) from the current study divided by the slope of the linear relationship (r = 0.479, n = 20, P < 0.02) between lens weight and age (11.48 years per meter) from (53).

Correlations
There was a strong linear correlation between the percentage of lens sphingolipid and lens lipid hydrocarbon chain order measured at 34.8°C (Fig. 5A, r = 0.8911, P < 0.01) until about 60% sphingolipid, above which there was little change in the hydrocarbon chain order. The percentage of lens sphingolipid correlated with lens lipid phase transition temperature (Fig. 5B, r = 0.7241, P < 0.01). The expected maximal lifespan of the whale was the longest of the species measured in this study and the percentage of whale lens sphingolipid fit well in the correlation between the percentage of lens sphingolipid and expected maximal lifespan measured in other studies (Fig. 5C, r = 0.8980, P < 0.01). The percentage of whale lens PC fit well in the correlation between the percentage of lens sphingolipid and expected maximal lifespan measured from other studies (Fig. 5D, r = 0.9141, P < 0.01). The percentage of DHSM was linearly related to total body length ( Table 1, r = 0.9942, P < 0.01), the age of the whale (51,52,54), and estimated age (Fig. 6, r = 1, P < 0.001). Lipid order and the phase transition temperature were also linearly related (r = 0.8911, P < 0.01).

DISCUSSION
Most whale species have well developed eyes. Underwater and aerial vision is critical for foraging at various dive depths, predator avoidance, navigation, and recognition of other individuals. Bowhead whales are the only baleen whale native to the Arctic. They have a small palpebral fissure and a thick and fleshy palpebra with a thick overlaying epidermis (Fig. 7 (75)). Compared with terrestrial mammals, the eyes of whales are well adapted to their light  limited aquatic environment with a thick cornea, sclera, and choroid that protect their eyes from the mechanical stress of diving and the cold temperature of the arctic water (76). The reflective layer of tapetum lucidum almost completely covers the fundus, enabling whales to see in low light conditions and sense light scattered from plankton (77). In addition, adaptation of retinal pigments further enhances their ability to see under low light conditions (78). The lens of the bowhead whale is very important for focusing light onto the retina because, unlike terrestrial mammals, the cornea of the bowhead whale is not refractive in water and of little optical importance (75). The center of the large spherical lens is in the center of the eyecup so that light coming from any direction is focused on the retina (77). Although zonules connect the lens to the ciliary process, the bowhead whale eye has no ciliary muscles (75), so accommodation is accomplished by the displacement of the lens by changes in intraocular pressure (77). Important to this study is the fact that bowhead whales do not get cataracts (54). The longer animals live, the longer they require clear lenses. One may ask the question, why do rats get cataracts at 2 years, dogs at 8 years, and humans at 60 years? Environmental causes (e.g., UV radiation, smoking) could contribute to some of the differences, however, ageing is the primary reason behind cataract development in various species. All of the species in Fig. 5C have diverse diets, optical water conditions, UV exposure, different accommodation processes, and other factors that could contribute to the variations observed in the figure; yet, the correlation between lens sphingolipid and lifespan is still significant. The phospholipid composition of adult lenses was reported in Fig. 5C. As discussed in the Introduction, with the progression of age, lens membranes of the species studied contain higher amounts of DHSM and lower amounts of PCs. In this study, we found this to be true of whales (Fig. 6) and their lens composition fits the curves in Fig. 5. Is this coincidental or does it have a scientific basis?
We found that whale lenses have one of the highest sphingolipid contents of lenses from the species studied. Lens membranes with a high content of saturated sphingolipids, like bowhead whale lenses, are less susceptible to oxidation because there is relatively less oxygen in these ordered bilayers, as well as fewer double bonds to become oxidized (15,(79)(80)(81)(82)(83). In this study, we found that whale lens lipid composition and structure support the finding that lens lipid hydrocarbon order is directly related to the sphingolipid content and indirectly related to the PC content of the lenses of many animals (Fig. 5). Thus for the whale lens, a high saturated sphingolipid content and increased head group interactions cause the lipid hydrocarbon chain region to become more ordered (63,64). As a consequence of higher lipid order, membranes may be less susceptible to oxidative damage because oxygen is five times more soluble in lipid membranes than it is in water (84)(85)(86)(87)(88)(89)(90). In addition, oxygen is five to ten times more soluble in fluid membranes (90), such as membranes low in sphingolipids, than it is in water.
Bowhead whale lenses have one of the highest levels of cholesterol measured for lenses. Above a molar cholesterol/phospholipid ratio of about two, cholesterol crystalline bilayer domains form (91,92). Because the phospholipid domain of the whale lens membranes is saturated with cholesterol, "the physical properties of the phospholipid bilayer portion of lens lipid membranes constant and independent of changes in the PL [phospholipid] composition that occur with age" (91, p. 722). While cholesterol probably plays a minor role in determining lens membrane phospholipid structure (71,91), it is likely to play a role in raft formation (93) and protein aggregation. One purpose of cholesterol in the lens may be to antagonize the binding of -crystallin to lens membranes (94). It has been proposed that -crystallin binding to lens membranes may serve as a "crystallization seed" for the binding of other proteins to the membrane, resulting in protein aggregation and light scattering (95). Thus, a high level of cholesterol in whale lenses could inhibit protein aggregation and cataract in whale lenses. It should be noted that for intact lens membranes, "… this beneficial effect of cholesterol on light scattering is compensated by the effect of integral membrane proteins" (91, p. 721). A high amount of cholesterol in the whale lens may also be important because cholesterol causes the lens membranes to be less permeable to oxygen, which may serve to keep oxygen in the outer regions of the lens long enough for the mitochondria to degrade it (96,97).
The high dihydrosphingolipid to sphingolipid ratio, 100%, in the oldest whale lenses is similar to that for human lenses (80%, Ref. 98) and may inhibit lens growth. It  Table 3. An increase in the stretching frequency is associated with an increase in hydrocarbon chain trans rotomers, disorder, and fluidity. In summary, the long-lived bowhead whale exhibits a variety of lens adaptations that confer resistance to oxidation, thereby allowing these membranes to stay clear for a relatively longer time than is the case in many other species. The specialized lens chemical composition in combination with an adapted external eye anatomy provide the bowhead Data recalculated from previous studies: rabbit (70), bovine (71), rat and camel (35), guinea pig (72), and humans (8,73,74). Coop, cooperativity; Max, maximum; Min, minimum; Tc, phase transition temperature. whales the evolutionary anatomical-physiological tools to protect and maintain vision for a long life under extreme arctic environmental conditions. The strong correlation between sphingolipid and lifespan may form a basis for future studies, which are needed because correlations do not infer cause. One could hope that if human lenses could be made to have a lipid composition similar to whales, like the bowhead whale, humans would not develop age-related cataracts for over 100 years. Fig. 6. Relationship between bowhead whale lens dihydrosphingomyelin content and estimated age. From Table 1. Linear curve fit, 3rd order (r = 1) (solid black line).